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Sample records for membrane androgen binding

  1. The effect of anabolic-androgenic steroids on aromatase activity and androgen receptor binding in the rat preoptic area.

    PubMed

    Roselli, C E

    1998-05-11

    The level of aromatase in the preoptic area of rats is transcriptionally regulated through a specific androgen-receptor mediated mechanism and can be used as a measure of central androgenic effect. Therefore, several commonly abused anabolic-androgenic steroids (AAS) were tested for their ability to induce aromatase activity in the preoptic area of castrated rats. In addition, we determined the relative binding affinities of these compounds for the androgen receptor, as well as their ability to bind androgen receptor in vivo following subcutaneous injections. All of the AAS compounds tested significantly stimulated POA aromatase activity above castrate levels. The compounds that produced the greatest stimulation of aromatase activity were those that bound most avidly to the androgen receptor in vitro (i.e., testosterone, dihydrotestosterone and nandrolone). In contrast, the 17alpha-alkylated compounds that were tested (stanozolol, danazol, methandrostenolone) modestly stimulated aromatase and were weak competitors for the androgen receptor. The subcutaneous injection of AAS compounds increased the concentrations of occupied nuclear androgen receptors in the brain, but the magnitude of effect was not related to their potency for inducing aromatase or their relative binding affinity for the androgen receptor suggesting that androgen receptor occupancy in POA is not correlated with the action of androgen on aromatase. The present results help explain the behavioral effects of AAS compounds in rats. PMID:9593936

  2. Binding kinetics and physical properties of androgen receptor in androgen-dependent Shionogi mammary carcinoma 115.

    PubMed

    Nohno, T

    1981-02-01

    The characteristics of the androgen receptor in the cytoplasmic fraction of Shionogi carcinoma 115 were studied in vitro by means of charcoal adsorption assay, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis. The equilibrium dissociation constant for [3H]dihydrotestosterone (Kd = (2-5) X 10(-11) M) was estimated from independently determined rates of association and dissociation, and was lower by one order of magnitude than the value obtained by saturation analysis (Kd = (2-8) X 10(-10) M). Evaluation of the effect of temperature on receptor binding of androgen allowed the estimation of several thermodynamic parameters, including activation energies of association (4 kcal/mol) and dissociation (14 kcal/mol), the apparent free energy (-13 kcal/mol), enthalpy (-9 kcal/mol), and entropy (+14 cal/mol per K). The receptor was greatly stabilized when bound with androgen. The results indicate how the lability of the unbound receptor and slow rate of dihydrotestosterone-receptor interaction can influence the estimation of dissociation constants by usual saturation analysis. The sedimentation coefficient of androgen receptor in freshly prepared cytosol was 6S, and became 7S after storage for 2 months at -80 degrees C. The 7S conversion of the receptor was reversed by treatment with heparin. In all cases, a single 5S peak was obtained in the presence of 0.5 M KCl. On electrophoresis in heparin-containing polyacrylamide gel, protein-bound radioactive androgen migrated as a single peak (Rf = 0.5 in 5% gel). Differences in reported values for the sedimentation coefficient of androgen receptor in cytosol of Shionogi carcinoma 115 appear to be derived from aggregation of the receptor protein during the assay procedure. PMID:7240130

  3. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    EPA Science Inventory

    Rainbow Trout Androgen Receptor Alpha And Human Androgen Receptor: Comparisons in the COS Whole Cell Binding Assay
    Mary C. Cardon, L. Earl Gray, Jr. and Vickie S. Wilson
    U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle...

  4. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    EPA Science Inventory

    RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY.
    MC Cardon, PC Hartig,LE Gray, Jr. and VS Wilson.
    U.S. EPA, ORD, NHEERL, RTD, Research Triangle Park, NC, USA.
    Typically, in vitro hazard assessments for ...

  5. Metformin Inhibits Androgen-Induced IGF-IR Up-Regulation in Prostate Cancer Cells by Disrupting Membrane-Initiated Androgen Signaling

    PubMed Central

    Malaguarnera, Roberta; Sacco, Antonella; Morcavallo, Alaide; Squatrito, Sebastiano; Migliaccio, Antimo; Morrione, Andrea; Maggiolini, Marcello

    2014-01-01

    We have previously demonstrated that, in prostate cancer cells, androgens up-regulate IGF-I receptor (IGF-IR) by inducing cAMP-response element-binding protein (CREB) activation and CREB-dependent IGF-IR gene transcription through androgen receptor (AR)-dependent membrane-initiated effects. This IGF-IR up-regulation is not blocked by classical antiandrogens and sensitizes cells to IGF-I-induced biological effects. Metformin exerts complex antitumoral functions in various models and may inhibit CREB activation in hepatocytes. We, therefore, evaluated whether metformin may affect androgen-dependent IGF-IR up-regulation. In the AR+ LNCaP prostate cancer cells, we found that metformin inhibits androgen-induced CRE activity and IGF-IR gene transcription. CRE activity requires the formation of a CREB-CREB binding protein-CREB regulated transcription coactivator 2 (CRTC2) complex, which follows Ser133-CREB phosphorylation. Metformin inhibited Ser133-CREB phosphorylation and induced nuclear exclusion of CREB cofactor CRTC2, thus dissociating the CREB-CREB binding protein-CRTC2 complex and blocking its transcriptional activity. Similarly to metformin action, CRTC2 silencing inhibited IGF-IR promoter activity. Moreover, metformin blocked membrane-initiated signals of AR to the mammalian target of rapamycin/p70S6Kinase pathway by inhibiting AR phosphorylation and its association with c-Src. AMPK signals were also involved to some extent. By inhibiting androgen-dependent IGF-IR up-regulation, metformin reduced IGF-I-mediated proliferation of LNCaP cells. These results indicate that, in prostate cancer cells, metformin inhibits IGF-I-mediated biological effects by disrupting membrane-initiated AR action responsible for IGF-IR up-regulation and suggest that metformin could represent a useful adjunct to the classical antiandrogen therapy. PMID:24437490

  6. Selective polyamine-binding proteins. Spermine binding by an androgen-sensitive phosphoprotein.

    PubMed

    Liang, T; Mezzetti, G; Chen, C; Liao, S

    1978-09-01

    Rat ventral prostate contains an acidic protein which can bind spermine selectively. The relative binding affinities of various aliphatic amines for the protein are, in decreasing order, spermine greater than thermine greater than greater than putrecine greater than 1,10-diaminodecane, cadaverine and 1,12-diaminododecane. The binding protein has an isoelectric point at pH 4.3 and a sedimentation coefficient of 3 S. Its molecular weight is approx. 30 000. Histones and nuclear chromatin preparations of the prostate can interact with the binding protein. The spermine-binding activity of the purified prostate protein can be inactivated by treatment with intestinal alkaline phosphatases. The phosphatase treated preparation can then be reactivated by beef heart protein kinase in the presence of cyclic AMP and ATP. The spermine-binding activity of the prostate cytosol protein fraction decreases after castration, but increases very rapidly after the castrated rats are injected with 5alpha-dihydrotestosterone. This finding raises the possibility that, in the postate, certain androgen actions may be dependent on the androgen-induced increase in the acidic protein binding of polyamines and their translocation to a functional cellular site such as nuclear chromatin. In the prostate cytosol, spermine also binds to 4-S tRNAs and to a unique RNA which has a sedimentation coefficient of 1.5 S. PMID:28786

  7. Recognition and Accommodation at the Androgen Receptor Coactivator Binding Interface

    PubMed Central

    Hur, Eugene; Pfaff, Samuel J; Payne, E. Sturgis; Grøn, Hanne; Buehrer, Benjamin M

    2004-01-01

    Prostate cancer is a leading killer of men in the industrialized world. Underlying this disease is the aberrant action of the androgen receptor (AR). AR is distinguished from other nuclear receptors in that after hormone binding, it preferentially responds to a specialized set of coactivators bearing aromatic-rich motifs, while responding poorly to coactivators bearing the leucine-rich “NR box” motifs favored by other nuclear receptors. Under normal conditions, interactions with these AR-specific coactivators through aromatic-rich motifs underlie targeted gene transcription. However, during prostate cancer, abnormal association with such coactivators, as well as with coactivators containing canonical leucine-rich motifs, promotes disease progression. To understand the paradox of this unusual selectivity, we have derived a complete set of peptide motifs that interact with AR using phage display. Binding affinities were measured for a selected set of these peptides and their interactions with AR determined by X-ray crystallography. Structures of AR in complex with FxxLF, LxxLL, FxxLW, WxxLF, WxxVW, FxxFF, and FxxYF motifs reveal a changing surface of the AR coactivator binding interface that permits accommodation of both AR-specific aromatic-rich motifs and canonical leucine-rich motifs. Induced fit provides perfect mating of the motifs representing the known family of AR coactivators and suggests a framework for the design of AR coactivator antagonists. PMID:15328534

  8. Androgens.

    PubMed

    Iyer, Rakesh; Handelsman, David J

    2016-01-01

    Androgen abuse is the most potent and prevalent form of sports doping detected. It originated from the early years of the Cold War as an epidemic confined to drug cheating within elite power sports. In the decades following the end of the Cold War, it has become disseminated into an endemic based within the illicit drug subcultures serving recreational abusers seeking cosmetic body sculpting effects. Within sports, both direct androgen abuse (administration of androgens), as well as indirect androgen abuse (administration of nonandrogenic drugs to increase endogenous testosterone), is mostly readily detectable with mass spectrometry-based anti-doping urine tests. The ongoing temptation of fame and fortune and the effectiveness of androgen abuse in power sports continue to entice cheating via renewed approaches aiming to exploit androgens. These require ongoing vigilance, inventiveness in anti-doping science, and targeting coaches as well as athletes in order to build resilience against doping and maintain fairness in elite sport. The challenge of androgen abuse in the community among recreational abusers has barely been recognized and effective approaches remain to be developed. PMID:27347677

  9. Substitution of synthetic chimpanzee androgen receptor for human androgen receptor in competitive binding and transcriptional activation assays for EDC screening

    EPA Science Inventory

    The potential effect of receptor-mediated endocrine modulators across species is of increasing concern. In attempts to address these concerns we are developing androgen and estrogen receptor binding assays using recombinant hormone receptors from a number of species across differ...

  10. In Silico and In Vitro Investigation of the Piperine's Male Contraceptive Effect: Docking and Molecular Dynamics Simulation Studies in Androgen-Binding Protein and Androgen Receptor.

    PubMed

    Chinta, Gopichand; Ramya Chandar Charles, Mariasoosai; Klopčič, Ivana; Sollner Dolenc, Marija; Periyasamy, Latha; Selvaraj Coumar, Mohane

    2015-07-01

    Understanding the molecular mechanism of action of traditional medicines is an important step towards developing marketable drugs from them. Piperine, an active constituent present in the Piper species, is used extensively in Ayurvedic medicines (practiced on the Indian subcontinent). Among others, piperine is known to possess a male contraceptive effect; however, the molecular mechanism of action for this effect is not very clear. In this regard, detailed docking and molecular dynamics simulation studies of piperine with the androgen-binding protein and androgen receptors were carried out. Androgen receptors control male sexual behavior and fertility, while the androgen-binding protein binds testosterone and maintains its concentration at optimal levels to stimulate spermatogenesis in the testis. It was found that piperine docks to the androgen-binding protein, similar to dihydrotestosterone, and to androgen receptors, similar to cyproterone acetate (antagonist). Also, the piperine-androgen-binding protein and piperine-androgen receptors interactions were found to be stable throughout 30 ns of molecular dynamics simulation. Further, two independent simulations for 10 ns each also confirmed the stability of these interactions. Detailed analysis of the piperine-androgen-binding protein interactions shows that piperine interacts with Ser42 of the androgen-binding protein and could block the binding with its natural ligands dihydrotestosterone/testosterone. Moreover, piperine interacts with Thr577 of the androgen receptors in a manner similar to the antagonist cyproterone acetate. Based on the in silico results, piperine was tested in the MDA-kb2 cell line using the luciferase reporter gene assay and was found to antagonize the effect of dihydrotestosterone at nanomolar concentrations. Further detailed biochemical experiments could help to develop piperine as an effective male contraceptive agent in the future. PMID:26039262

  11. CCAAT/Enhancer binding protein β controls androgen-deprivation-induced senescence in prostate cancer cells.

    PubMed

    Barakat, D J; Zhang, J; Barberi, T; Denmeade, S R; Friedman, A D; Paz-Priel, I

    2015-11-26

    The processes associated with transition to castration-resistant prostate cancer (PC) growth are not well understood. Cellular senescence is a stable cell cycle arrest that occurs in response to sublethal stress. It is often overcome in malignant transformation to confer a survival advantage. CCAAT/Enhancer Binding Protein (C/EBP) β function is frequently deregulated in human malignancies and interestingly, androgen-sensitive PC cells express primarily the liver-enriched inhibitory protein isoform. We found that C/EBPβ expression is negatively regulated by androgen receptor (AR) activity and that treatment of androgen-sensitive cell lines with anti-androgens increases C/EBPβ mRNA and protein levels. Accordingly, we also find that C/EBPβ levels are significantly elevated in primary PC samples from castration-resistant compared with therapy-naive patients. Chromatin immunoprecipitation demonstrated enhanced binding of the AR to the proximal promoter of the CEBPB gene in the presence of dihydroxytestosterone. Upon androgen deprivation, induction of C/EBPβ is facilitated by active transcription as evident by increased histone 3 acetylation at the C/EBPβ promoter. Also, the androgen agonist R1881 suppresses the activity of a CEBPB promoter reporter. Loss of C/EBPβ expression prevents growth arrest following androgen deprivation or anti-androgen challenge. Accordingly, suppression of C/EBPβ under low androgen conditions results in reduced expression of senescence-associated secretory genes, significantly decreased number of cells displaying heterochromatin foci and increased numbers of Ki67-positive cells. Ectopic expression of C/EBPβ caused pronounced morphological changes, reduced PC cell growth and increased the number of senescent LNCaP cells. Lastly, we found that senescence contributes to PC cell survival under androgen deprivation, and C/EBPβ-deficient cells were significantly more susceptible to killing by cytotoxic chemotherapy following androgen

  12. Binding properties of androgen receptors. Evidence for identical receptors in rat testis, epididymis, and prostate.

    PubMed

    Wilson, E M; French, F S

    1976-09-25

    Androgen receptors in crude and partially purified 105,000 X g supernatant fractions from rat testis, epididymis, and prostate were studied in vitro using a charcoal adsorption assay and sucrose gradient centrifugation. Androgen metabolism was eliminated during receptor purification allowing determination of the kinetics of [3H]-androgen-receptor complex formation. In all three tissues, receptors were found to have essentially identical capabilities to bind androgen, with the affinity for [3H] dihydrotestosterone being somewhat higher than for [3H] testosterone. Equilibrium dissociation constants for [3H] dihydrotestosterone and [3H] testosterone (KD = 2 to 5 X 10(-10) M) were estimated from independently determined rates of association (ka congruent to 6 X 10(7) M-1 h-1 for [3H] dihydrotestosterone and 2 X 10(8) M-1 h-1 for [3H] testosterone) and dissociation (t 1/2 congruent to 40 hr for [3H] dihydrotestosterone and 15 h [3H] testosterone). Evaluation of the effect of temperature on androgen receptor binding of [3H]testosterone allowed estimation of several thermodynamic parameters, including activation energies of association and dissociation (delta H congruent to 14 kcal/mol), the apparent free energy (delta G congruent to -12 kcal/mol), enthalpy (delta H congruent to -2.5 kcal/mol), and entropy (delta S congruent to 35 cal col-1 K-1). Optimum receptor binding occurred at a pH of 8. Receptor stability was greatly enhanced when bound with androgen. Receptor specificity for testosterone and dihydrotestosterone was demonstrated by competitive binding assays. The potent synthetic androgen, 7 alpha, 17 alpha-dimethyl-19-nortestosterone, inhibited binding of [3H] testosterone or [3H] dihydrotesterone nearly as well as testosterone and dihydrotestosterone while larger amounts of 5 alpha-androstane-3alpha, 17 beta-diol and nonandrogenic steroids were required. Sedimentation coefficients of androgen receptors in all unfractionated supernatants were 4 and 5 to 8 S

  13. Increased cytosolic androgen receptor binding in rat striated muscle following denervation and disuse

    NASA Technical Reports Server (NTRS)

    Bernard, P. A.; Fishman, P. S.; Max, S. R.; Rance, N. E.

    1984-01-01

    The effects of denervation and disuse on cytosolic androgen receptor binding by rat striated muscle are investigated. Denervation of the extensor digitorum longus and tibialis anterior muscles caused by a 40-50-percent increase in cytosolic androgen receptor concentration with no change in apparent binding affinity. This effect was evident at 6 h postdenervation, maximal at 24 h, and declined to 120 percent of the control level 72 h after denervation. A 40-percent increase in cytosolic androgen receptor concentration was also noted 24 hr after denervation of the hormone-sensitive levator ani muscle. The effect of denervation on androgen receptors was blocked by in vivo injection of cycloheximide; therefore, de novo receptor synthesis probably is not involved in the observed increase. Disuse, produced by subperineurial injection of tetrodotoxin into the tibial and common peroneal branches of the sciatic nerve, mimicked the effect of denervation on androgen receptor binding, suggesting that neuromuscular activity is important in regulation of receptor concentration. Possible mechanisms subserving this effect are discussed.

  14. The androgen-binding protein gene is expressed in male and female rat brain.

    PubMed

    Wang, Y M; Bayliss, D A; Millhorn, D E; Petrusz, P; Joseph, D R

    1990-12-01

    Extracellular androgen-binding proteins (ABP) are thought to modulate the regulatory functions of androgens and the trans-acting nuclear androgen receptor. Testicular ABP and plasma sex hormone-binding globulin (SHBG), which is produced in liver, are encoded by the same gene. We have now found that the ABP-SHBG gene is also expressed in male and female rat brain. Immunoreactive ABP was found to be present in neuronal cell bodies throughout the brain as well as in fibers of the hypothalamic median eminence. The highest concentrations of immunoreactive cell bodies were located in the supraoptic and paraventricular nuclei. Likewise, ABP mRNA was present in all brain regions examined. Analysis of cDNA clones representing brain ABP mRNAs revealed amino acid sequence differences in brain and testicular ABPs. The protein encoded by an alternatively processed RNA has sequence characteristics suggesting that the protein could act as a competitior of ABP binding to cell surface receptors. These data and gene-sequencing experiments indicate that a specific ABP gene promoter is used for transcription initiation in brain. ABP may function in brain as an androgen carrier protein; however, in view of the widespread presence of ABP and ABP mRNA in brain, the protein may have a much broader, yet unknown, function. PMID:1701136

  15. Detection of persistent organic pollutants binding modes with androgen receptor ligand binding domain by docking and molecular dynamics

    PubMed Central

    2013-01-01

    Background Persistent organic pollutants (POPs) are persistent in the environment after release from industrial compounds, combustion productions or pesticides. The exposure of POPs has been related to various reproductive disturbances, such as reduced semen quality, testicular cancer, and imbalanced sex ratio. Among POPs, dichlorodiphenyldichloroethylene (4,4’-DDE) and polychlorinated biphenyls (PCBs) are the most widespread and well-studied compounds. Recent studies have revealed that 4,4’-DDE is an antagonist of androgen receptor (AR). However, the mechanism of the inhibition remains elusive. CB-153 is the most common congener of PCBs, while the action of CB-153 on AR is still under debate. Results Molecular docking and molecular dynamics (MD) approaches have been employed to study binding modes and inhibition mechanism of 4,4’-DDE and CB-153 against AR ligand binding domain (LBD). Several potential binding sites have been detected and analyzed. One possible binding site is the same binding site of AR natural ligand androgen 5α-dihydrotestosterone (DHT). Another one is on the ligand-dependent transcriptional activation function (AF2) region, which is crucial for the co-activators recruitment. Besides, a novel possible binding site was observed for POPs with low binding free energy with the receptor. Detailed interactions between ligands and the receptor have been represented. The disrupting mechanism of POPs against AR has also been discussed. Conclusions POPs disrupt the function of AR through binding to three possible biding sites on AR/LBD. One of them shares the same binding site of natural ligand of AR. Another one is on AF2 region. The third one is in a cleft near N-terminal of the receptor. Significantly, values of binding free energy of POPs with AR/LBD are comparable to that of natural ligand androgen DHT. PMID:24053684

  16. Opposing effects of estradiol- and testosterone-membrane binding sites on T47D breast cancer cell apoptosis

    SciTech Connect

    Kampa, Marilena; Nifli, Artemissia-Phoebe; Charalampopoulos, Ioannis; Alexaki, Vassilia-Ismini; Theodoropoulos, Panayiotis A.; Stathopoulos, Efstathios N.; Gravanis, Achille; Castanas, Elias . E-mail: castanas@med.uoc.gr

    2005-07-01

    Classical steroid mode of action involves binding to intracellular receptors, the later acting as ligand-activated nuclear transcription factors. Recently, membrane sites for different steroids have been also identified, mediating rapid, non-genomic, steroid actions. Membrane sites for estrogen and androgen have been found in a number of different cell types, bearing or not classical intracellular receptors. In the present study, with the use of radioligand binding, flow cytometry and confocal laser microscopy, we report that T47D human breast cancer cells express specific and saturable membrane receptors for both estrogen (K {sub D} 4.06 {+-} 3.31 nM) and androgen (K {sub D} 7.64 {+-} 3.15 nM). Upon activation with BSA-conjugated, non-permeable ligands (E{sub 2}-BSA and testosterone-BSA), membrane estrogen receptors protect cells from serum-deprivation-induced apoptosis, while androgen receptors induce apoptosis in serum-supplemented T47D cells. In addition, co-incubation of cells with a fixed concentration of one steroid and varying concentrations of the other reversed the abovementioned effect (apoptosis for androgen, and anti-apoptosis for E{sub 2}), suggesting that the fate of the cell depends on the relative concentration of either steroid in the culture medium. We also report the identification of membrane receptors for E{sub 2} and androgen in biopsy slides from breast cancer patients. Both sites are expressed, with the staining for membrane E{sub 2} being strongly present in ER-negative, less differentiated, more aggressive tumors. These findings suggest that aromatase inhibitors may exert their beneficial effects on breast cancer by also propagating the metabolism of local steroids towards androgen, inducing thus cell apoptosis through membrane androgen receptor activation.

  17. Selectivity in progesterone and androgen receptor binding of progestagens used in oral contraceptives

    SciTech Connect

    Kloosterboer, H.J.; Vonk-Noordegraaf, C.A.; Turpijn, E.W.

    1988-09-01

    The relative binding affinities (RBAs) of four progestational compounds (norethisterone, levonorgestrel, 3-keto-desogestrel and gestodene) for the human progesterone and androgen receptors were measured in MCF-7 cytosol and intact MCF-7 cells. For the binding to the progesterone receptor, both Org 2058 and Org 3236 (or 3-keto-desogestrel) were used as labelled ligands. The following ranking (low to high) for the RBA of the nuclear (intact cells) progesterone receptor irrespective of the ligand used is found: norethisterone much less than levonorgestrel less than 3-keto-destogestrel less than gestodene. The difference between the various progestagens is significant with the exception of that between 3-keto-desogestrel and gestodene, when Org 2058 is used as ligand. For the cytosolic progesterone receptor, the same order is found with the exception that similar RBAs are found for gestodene and 3-keto-desogestrel. The four progestagens clearly differ with respect to binding to the androgen receptor using dihydrotestosterone as labelled ligand in intact cells; the ranking (low to high) is: norethisterone less than 3 keto-desogestrel less than levonorgestrel and gestodene. The difference between 3-keto-desogestrel and levonorgestrel or gestodene is significant. The selectivity indices (ratio of the mean RBA for the progesterone receptor to that of androgen receptor) in intact cells are significantly higher for 3-keto-desogestrel and gestodene than for levonorgestrel and norethisterone. From these results we conclude that the introduction of the 18-methyl in norethisterone (levonorgestel) increases both the binding to the progesterone and androgen receptors.

  18. Androgen insensitivity.

    PubMed

    Gottlieb, B; Pinsky, L; Beitel, L K; Trifiro, M

    1999-12-29

    The androgen receptor (AR) protein regulates transcription of certain genes. Usually, this activity depends upon a central DNA-binding domain that permits the binding of androgen-AR complexes to regulatory DNA sequences near or in a target gene. The AR also has a C-terminal androgen-binding domain (ABD) and an N-terminal modulatory domain. These domains interact among themselves and with coregulatory, nonreceptor proteins to determine vector control over a gene's transcription rate. The precise roles of these proteins are active research areas. Severe X-linked androgen receptor gene (AR) mutations cause complete androgen insensitivity, mild ones impair virilization with or without infertility, and moderate ones sometimes yield a wide phenotypic spectrum among sibs. Different expressivity may reflect variability of AR-interactive proteins. The family history must identify heterozygous XX females with sparse, delayed, or asymmetric pubic/axillary hair or delayed menarche and infertile XY maternal aunts or uncles. Mutation type and density vary along the length of the AR. N-terminal polyglutamine tract expansion limits AR transactivation, causing a form of mild androgen insensitivity. Analysis of ABD mutations that do not impair androgen binding or impair it selectively will illuminate its intradomain properties. For partial androgen insensitivity and mild androgen insensitivity, pharmacotherapy with certain androgens or other steroids may overcome some dysfunction of certain mutant ARs. Experience with this approach is limited; outcomes have been generally disappointing. PMID:10727996

  19. Secretin: specific binding to rat brain membranes

    SciTech Connect

    Fremeau, R.T. Jr.; Jensen, R.T.; Charlton, C.G.; Miller, R.L.; O'Donohue, T.L.; Moody, T.W.

    1983-08-01

    The binding of (/sup 125/I)secretin to rat brain membranes was investigated. Radiolabeled secretin bound with high affinity (KD . 0.2 nM) to a single class of noninteracting sites. Binding was specific, saturable, and reversible. Regional distribution studies indicated that the specific binding was greatest in the cerebellum, intermediate in the cortex, thalamus, striatum, hippocampus, and hypothalamus, and lowest in the midbrain and medulla/pons. Pharmacological studies indicated that only secretin, but not other peptides, inhibits binding of (/sup 125/I)secretin with high affinity. Also, certain guanine nucleotides inhibited high affinity binding. These data indicate that rat brain membranes possess high affinity binding sites specific for secretin and that with the use of (/sup 125/I) secretin the kinetics, stoichiometry, specificity, and distribution of secretin receptors can be directly investigated.

  20. Androgen up-regulates vascular endothelial growth factor expression in prostate cancer cells via an Sp1 binding site

    PubMed Central

    2013-01-01

    Background Vascular Endothelial Growth Factor (VEGF) is regulated by a number of different factors, but the mechanism(s) behind androgen-mediated regulation of VEGF in prostate cancer are poorly understood. Results Three novel androgen receptor (AR) binding sites were discovered in the VEGF promoter and in vivo binding of AR to these sites was demonstrated by chromatin immunoprecipitation. Mutation of these sites attenuated activation of the VEGF promoter by the androgen analog, R1881 in prostate cancer cells. The transcription factors AR and Sp1 were shown to form a nuclear complex and both bound the VEGF core promoter in chromatin of hormone treated CWR22Rv1 prostate cancer cells. The importance of the Sp1 binding site in hormone mediated activation of VEGF expression was demonstrated by site directed mutagenesis. Mutation of a critical Sp1 binding site (Sp1.4) in the VEGF core promoter region prevented activation by androgen. Similarly, suppression of Sp1 binding by Mithramycin A treatment significantly reduced VEGF expression. Conclusions Our mechanistic study of androgen mediated induction of VEGF expression in prostate cancer cells revealed for the first time that this induction is mediated through the core promoter region and is dependent upon a critical Sp1 binding site. The importance of Sp1 binding suggests that therapy targeting the AR-Sp1 complex may dampen VEGF induced angiogenesis and, thereby, block prostate cancer progression, helping to maintain the indolent form of prostate cancer. PMID:23369005

  1. Structure of the ligand-binding domain (LBD) of human androgen receptor in complex with a selective modulator LGD2226

    SciTech Connect

    Wang, Feng; Liu, Xiao-qin; Li, He; Liang, Kai-ni; Miner, Jeffrey N.; Hong, Mei; Kallel, E. Adam; Oeveren, Arjan van; Zhi, Lin; Jiang, Tao

    2006-11-01

    Crystal structure of the ligand-binding domain of androgen receptor in complex with LGD2226. The androgen receptor (AR) is a ligand-inducible steroid hormone receptor that mediates androgen action, determining male sexual phenotypes and promoting spermatogenesis. As the androgens play a dominant role in male sexual development and function, steroidal androgen agonists have been used clinically for some years. However, there is a risk of potential side effects and most steroidal androgens cannot be dosed orally, which limits the use of these substances. 1,2-Dihydro-6-N,N-bis(2,2,2-trifluoroethyl) amino-4-trifluoromethyl-2-quinolinone (LGD2226) is a synthetic nonsteroidal ligand and a novel selective AR modulator. The crystal structure of the complex of LGD2226 with the androgen receptor ligand-binding domain (AR LBD) at 2.1 Å was solved and compared with the structure of the AR LBD–R1881 complex. It is hoped that this will aid in further explaining the selectivity of LGD2226 observed in in vitro and in vivo assays and in developing more selective and effective therapeutic agents.

  2. An androgenic gland membrane-anchored gene associated with the crustacean insulin-like androgenic gland hormone.

    PubMed

    Rosen, Ohad; Manor, Rivka; Weil, Simy; Aflalo, Eliahu D; Bakhrat, Anna; Abdu, Uri; Sagi, Amir

    2013-06-01

    Crustacean male sexual differentiation is governed by the androgenic gland (AG) and specifically by the secreted insulin-like AG hormone (IAG), thus far identified in several decapod species including the Australian red claw crayfish Cherax quadricarinatus (termed Cq-IAG). While a few insulin-like AG genes have been identified in crustaceans, other AG-specific genes have not been documented until now. In the present study, we describe the recent identification of a non-IAG AG-specific transcript obtained from the C. quadricarinatus AG cDNA library. This transcript, termed C. quadricarinatus membrane-anchored AG-specific factor (Cq-MAG), was fully sequenced and found to encode a putative product of 189 amino acids including a signal anchoring peptide. Expression of a recombinant GFP fusion protein lacking the signal anchor encoding sequence dramatically affected recombinant protein localization pattern. While the expression of the deleterious fusion protein was observed throughout most of the cell, the native GFP::Cq-MAG fusion protein was observed mainly surrounding the periphery of the nucleus, demonstrating an endoplasmic reticulum (ER)-like localization pattern. Moreover, co-expression of the wild-type Cq-MAG (fused to GFP) and the Cq-IAG hormone revealed that these peptides indeed co-localize. This study is the first to report a protein specifically associated with the insulin-like AG hormone in addition to the finding of another AG-specific transcript in crustaceans. Previous knowledge suggests that insulin/insulin-like factor secretion involves tissue-specific transcripts and membrane-anchored proteins. In this regard, Cq-MAG's tissue specificity, anchoring properties and intracellular co-localization with Cq-IAG suggest that it may play a role in the processing and secretion of this insulin-like AG hormone. PMID:23470660

  3. BA321, a novel carborane analog that binds to androgen and estrogen receptors, acts as a new selective androgen receptor modulator of bone in male mice.

    PubMed

    Watanabe, Kenta; Hirata, Michiko; Tominari, Tsukasa; Matsumoto, Chiho; Endo, Yasuyuki; Murphy, Gillian; Nagase, Hideaki; Inada, Masaki; Miyaura, Chisato

    2016-09-01

    Carboranes are a class of carbon-containing polyhedral boron cluster compounds with globular geometry and hydrophobic surface that interact with hormone receptors such as estrogen receptor (ER) and androgen receptor (AR). We have synthesized BA321, a novel carborane compound, which binds to AR. We found here that it also binds to ERs, ERα and ERβ. In orchidectomized (ORX) mice, femoral bone mass was markedly reduced due to androgen deficiency and BA321 restored bone loss in the male, whilst the decreased weight of seminal vesicle in ORX mice was not recovered by administration of BA321. In female mice, BA321 acts as a pure estrogen agonist, and restored both the loss of bone mass and uterine atrophy due to estrogen deficiency in ovariectomized (OVX) mice. In bone tissues, the trabecular bone loss occurred in both ORX and OVX mice, and BA321 completely restored the trabecular bone loss in both sexes. Cortical bone loss occurred in ORX mice but not in OVX mice, and BA321 clearly restored cortical bone loss due to androgen deficiency in ORX mice. Therefore, BA321 is a novel selective androgen receptor modulator (SARM) that may offer a new therapy option for osteoporosis in the male. PMID:27402268

  4. Androgen action.

    PubMed

    Werner, Ralf; Holterhus, Paul-Martin

    2014-01-01

    Androgens are important for male sex development and physiology. Their actions are mediated by the androgen receptor (AR), a ligand-dependent nuclear transcription factor. The activity of the AR is controlled at multiple stages due to ligand binding and induced structural changes assisted by the foldosome, compartmentalization, recruitment of coregulators, posttranslational modifications and chromatin remodeling, leading to subsequent transcription of androgen-responsive target genes. Beside these short-term androgen actions, there is phenomenological and experimental evidence of long-term androgen programming in mammals and in the human during sensitive programming time windows, both pre- and postnatally. At the molecular level, research into androgen insensitivity syndrome has unmasked androgen programming at the transcriptome level, in genital fibroblasts and peripheral blood mononuclear cells, and at the epigenome level. Androgens are crucial for male sex development and physiology during embryogenesis, at puberty and in adult life. Testosterone and its more potent metabolite, dihydrotestosterone, which is converted from testosterone within the target cell by 5α-reductase II, are the main androgens involved in male sex differentiation. Androgen action is mediated by a single AR. The AR belongs to the nuclear receptor 3 group C, composed of the glucocorticoid receptor (NR3C1), mineralocorticoid receptor (NR3C2), progesterone receptor (NR3C3) and AR (NR3C4), and acts as a ligand-dependent transcription factor. PMID:25247642

  5. Single amino acid substitutions at 2 of 14 positions in an ultra-conserved region of the androgen receptor yield an androgen-binding domain that is reversibly thermolabile

    SciTech Connect

    Vasiliou, M.; Lumbroso, R.; Alvarado, C.

    1994-09-01

    The stereochemistry of the androgen receptor (AR) that is responsible for androgen-specific binding and for its contribution to the transregulatory attributes of an androgen-receptor complex are unknown. Our objective is to define structure-function relations of the human AR by correlating germline missense mutations at its X-linked locus with its resultant misbehavior. Subjects with Arg773Cys have complete androgen insensitivity. We and several other laboratories have reported that their genital skin fibroblasts (GSF) have negligible androgen-binding activity at 37{degrees}. We have found that Phe763Leu also causes CAI, but with approximately 10 fmol/mg protein androgen-binding activity at 37{degrees} (R-deficient). Within COS-1 cells transfected with each mutant AR cDNA, Phe763Leu and Arg773Cys androgen-binding activities are reversibly thermolabile, by a factor of 2, at 37{degrees} versus 22{degrees}, only in the presence of androgen; in the absence of androgen they are thermostable at 37{degrees}. We have discovered that (for a reason yet unknown) the GSF from a third family with Arg773Cys (and no other coding sequence mutation) have 20-40 mol/mg protein of androgen-binding activity at 37{degrees} when measured with 3-6 nFM androgen. This activity reversibly doubles at 22{degrees}. The reversible thermolability of an AR with Arg773Cys (and probably with Phe763Leu) is demonstrable within GSF. Ligand-dependence of this thermolability implies that ligand induces these mutant AR to undergo a deviant conformational change in, or near, a 14-aa region that shares 90% identity/similarity with its closest receptor relatives.

  6. Identification and characterization of membrane androgen receptors in the ZIP9 zinc transporter subfamily: I. Discovery in female atlantic croaker and evidence ZIP9 mediates testosterone-induced apoptosis of ovarian follicle cells.

    PubMed

    Berg, A Håkan; Rice, Charles D; Rahman, Md Saydur; Dong, Jing; Thomas, Peter

    2014-11-01

    Rapid, cell surface-initiated, pregenomic androgen actions have been described in various vertebrate cells, but the receptors mediating these actions remain unidentified. We report here the cloning and expression of a cDNA from Atlantic croaker (Micropogonias undulatus) ovaries encoding a 33-kDa, seven-transmembrane protein with binding and signaling characteristics of a membrane androgen receptor that is unrelated to any previously described steroid receptor. Instead, croaker membrane androgen receptor has 81-93% amino acid sequence identity with zinc transporter ZIP9 (SLC39A9) subfamily members, indicating it is a ZIP9 protein. Croaker ZIP9 is expressed in gonadal tissues and in brain and is up-regulated in the ovary by reproductive hormones. Croaker ZIP9 protein is localized to plasma membranes of croaker granulosa cells and human breast cancer (SKBR-3) cells stably transfected with ZIP9. Recombinant croaker ZIP9 has a high affinity (dissociation constant, Kd, 12.7 nM), limited capacity (maximal binding capacity 2.8 nM/mg protein), displaceable, single binding site-specific for androgens, characteristic of steroid receptors. Testosterone activates a stimulatory G protein coupled to ZIP9, resulting in increased cAMP production. Testosterone promotes serum starvation-induced cell death and apoptosis in transfected cells and in croaker ovarian follicle cells that is associated with rapid increases in intracellular free zinc concentrations, suggesting an involvement of zinc in this nonclassical androgen action to promote apoptosis. These responses to testosterone are abrogated by treatment with ZIP9 small interfering RNA. The results provide the first evidence that zinc transporter proteins can function as specific steroid membrane receptors and indicate a previously unrecognized signaling pathway mediated by steroid receptors involving alterations in intracellular zinc. PMID:25014354

  7. Association of androgen with gender difference in serum adipocyte fatty acid binding protein levels

    PubMed Central

    Hu, Xiang; Ma, Xiaojing; Pan, Xiaoping; Luo, Yuqi; Xu, Yiting; Xiong, Qin; Bao, Yuqian; Jia, Weiping

    2016-01-01

    Clinical investigations have indicated women have higher levels of adipocyte fatty acid binding protein (A-FABP) than men. The present study aimed to identify factors related to gender difference in serum A-FABP levels. A total of 507 participants (194 men, 132 premenopausal women, and 181 postmenopausal women) were enrolled in the present study. Serum A-FABP levels increased in the order from men to premenopausal women to postmenopausal women in both body mass index categories (<25.0 and ≥25.0 kg/m2; all P < 0.05). Multiple stepwise regression analyses showed that after adjustment for factors related to serum A-FABP levels, the trunk fat mass was an independent and positive factor of serum A-FABP levels. For men, total testosterone was associated independently and inversely with serum A-FABP levels. For pre- and postmenopausal women, bioavailable testosterone and total testosterone were independent and positive factors associated with serum A-FABP levels, respectively. The present study demonstrated that the androgen was correlated with the serum A-FABP levels negatively in men, but positively in women. With these effects on the fat content, especially trunk fat, androgen might contribute to the gender difference in serum A-FABP levels. PMID:27270834

  8. Association of androgen with gender difference in serum adipocyte fatty acid binding protein levels.

    PubMed

    Hu, Xiang; Ma, Xiaojing; Pan, Xiaoping; Luo, Yuqi; Xu, Yiting; Xiong, Qin; Bao, Yuqian; Jia, Weiping

    2016-01-01

    Clinical investigations have indicated women have higher levels of adipocyte fatty acid binding protein (A-FABP) than men. The present study aimed to identify factors related to gender difference in serum A-FABP levels. A total of 507 participants (194 men, 132 premenopausal women, and 181 postmenopausal women) were enrolled in the present study. Serum A-FABP levels increased in the order from men to premenopausal women to postmenopausal women in both body mass index categories (<25.0 and ≥25.0 kg/m(2); all P < 0.05). Multiple stepwise regression analyses showed that after adjustment for factors related to serum A-FABP levels, the trunk fat mass was an independent and positive factor of serum A-FABP levels. For men, total testosterone was associated independently and inversely with serum A-FABP levels. For pre- and postmenopausal women, bioavailable testosterone and total testosterone were independent and positive factors associated with serum A-FABP levels, respectively. The present study demonstrated that the androgen was correlated with the serum A-FABP levels negatively in men, but positively in women. With these effects on the fat content, especially trunk fat, androgen might contribute to the gender difference in serum A-FABP levels. PMID:27270834

  9. BINDING OF STEROIDS AND ENVIRONMENTAL CHEMICALS TO THE RAINBOW TROUT ANDROGEN RECEPTOR ALPHA EXPRESSED IN COS CELLS

    EPA Science Inventory

    Binding of Steroids and Environmental Chemicals to the Rainbow Trout Androgen Receptor Alpha Expressed in COS Cells.

    Mary C. Cardon, L. Earl Gray. Jr., Phillip C. Hartig and Vickie S. Wilson
    U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology...

  10. Testosterone regulates the autophagic clearance of androgen binding protein in rat Sertoli cells

    PubMed Central

    Ma, Yi; Yang, Hao-Zheng; Xu, Long-Mei; Huang, Yi-Ran; Dai, Hui-Li; Kang, Xiao-Nan

    2015-01-01

    Dysregulation of androgen-binding protein (ABP) is associated with a number of endocrine and andrology diseases. However, the ABP metabolism in Sertoli cells is largely unknown. We report that autophagy degrades ABP in rat Sertoli cells, and the autophagic clearance of ABP is regulated by testosterone, which prolongs the ABP biological half-life by inhibiting autophagy. Further studies identified that the autophagic clearance of ABP might be selectively regulated by testosterone, independent of stress (hypoxia)-induced autophagic degradation. These data demonstrate that testosterone up-regulates ABP expression at least partially by suppressing the autophagic degradation. We report a novel finding with respect to the mechanisms by which ABP is cleared, and by which the process is regulated in Sertoli cells. PMID:25745956

  11. Assessment of a recombinant androgen receptor binding assay: initial steps towards validation.

    PubMed

    Freyberger, Alexius; Weimer, Marc; Tran, Hoai-Son; Ahr, Hans-Jürgen

    2010-08-01

    Despite more than a decade of research in the field of endocrine active compounds with affinity for the androgen receptor (AR), still no validated recombinant AR binding assay is available, although recombinant AR can be obtained from several sources. With funding from the European Union (EU)-sponsored 6th framework project, ReProTect, we developed a model protocol for such an assay based on a simple AR binding assay recently developed at our institution. Important features of the protocol were the use of a rat recombinant fusion protein to thioredoxin containing both the hinge region and ligand binding domain (LBD) of the rat AR (which is identical to the human AR-LBD) and performance in a 96-well plate format. Besides two reference compounds [dihydrotestosterone (DHT), androstenedione] ten test compounds with different affinities for the AR [levonorgestrel, progesterone, prochloraz, 17alpha-methyltestosterone, flutamide, norethynodrel, o,p'-DDT, dibutylphthalate, vinclozolin, linuron] were used to explore the performance of the assay. At least three independent experiments per compound were performed. The AR binding properties of reference and test compounds were well detected, in terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using recombinant AR preparations. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.6. Our data demonstrate that the assay reliably ranked compounds with strong, weak, and no/marginal affinity for the AR with high accuracy. It avoids the manipulation and use of animals, as a recombinant protein is used and thus contributes to the 3R concept. On the whole, this assay is a promising candidate for further validation. PMID:19833195

  12. Identification and characterization of a prostate-specific androgen-independent protein-binding site in the probasin promoter.

    PubMed Central

    Yeung, Lillian H Y; Read, Jason T; Sorenson, Pernille; Nelson, Colleen C; Jia, William; Rennie, Paul S

    2003-01-01

    In this study we investigated the combination of transcription factors and proteins binding to the proximal part of the prostate-specific probasin (PB) promoter. Using DNaseI in vitro footprinting, several protected regions were identified on the proximal PB promoter (nucleotides -286 to +28 relative to the transcription start site) when nuclear extracts from LNCaP, a human prostate cancer cell line, were used. Four of the protected areas were observed only when LNCaP nuclear extracts treated with synthetic androgen (10 nM R1881) were used. Two other regions, referred to as FPI and FPII, showed protection regardless of the presence or absence of androgen. When DNaseI footprinting was done using other prostate and non-prostate nuclear extracts, protection of the FPII region was only seen in prostate cell lines. These androgen-independent regions were further tested for tissue and binding specificity using the electrophoretic mobility-shift assay. Eight complexes formed with the FPI probe while four complexes were observed with the FPII probe on incubation with the tested nuclear extracts. Methylation protection assays reveal that prostate cancer cell lines yield slightly different protection patterns for some of the protein complexes formed with non-prostate-derived cell lines, suggesting the presence of prostate-enriched or -exclusive proteins. Site-directed mutagenesis of the protected nucleotides within FPII resulted in a significant reduction in expression from the PB promoter. Identification of proteins binding to the FPII region revealed the participation of nuclear factor I (NF-I) or a closely related protein, although other unknown proteins are also involved. Defining the DNA and protein components that dictate prostate-specific expression of the PB promoter in an androgen-independent manner would provide a strong basis for the design and development of a gene therapy for systemic treatment of androgen-independent prostate cancer. PMID:12540291

  13. β-Catenin Binds to the Activation Function 2 Region of the Androgen Receptor and Modulates the Effects of the N-Terminal Domain and TIF2 on Ligand-Dependent Transcription

    PubMed Central

    Song, Liang-Nian; Herrell, Roger; Byers, Stephen; Shah, Salimuddin; Wilson, Elizabeth M.; Gelmann, Edward P.

    2003-01-01

    β-Catenin is a multifunctional molecule that is activated by signaling through WNT receptors. β-Catenin can also enhance the transcriptional activity of some steroid hormone receptors such as the androgen receptor and retinoic acid receptor α. Androgens can affect nuclear translocation of β-catenin and influence its subcellular distribution. Using mammalian two-hybrid binding assays, analysis of reporter gene transcription, and coimmunoprecipitation, we now show that β-catenin binds to the androgen receptor ligand-binding domain (LBD) and modulates the transcriptional effects of TIF2 and the androgen receptor N-terminal domain (NTD). In functional assays, β-catenin bound to androgen receptor only in the presence of ligand agonists, not antagonists. β-Catenin binding to the androgen receptor LBD was independent of and cooperative with the androgen receptor NTD and the p160 coactivator TIF2, both of which bind to the activation function 2 (AF-2) region of the androgen receptor. Different mutations of androgen receptor helix 3 amino acids disrupted binding of androgen receptor NTD and β-catenin. β-Catenin, androgen receptor NTD, and TIF2 binding to the androgen receptor LBD were affected similarly by a subset of helix 12 mutations, but disruption of two sites on helix 12 affected only binding of β-catenin and not of TIF2 or the androgen receptor NTD. Mutational disruption of each of five LXXLL peptide motifs in the β-catenin armadillo repeats did not disrupt either binding to androgen receptor or transcriptional coactivation. ICAT, an inhibitor of T-cell factor 4 (TCF-4), and E-cadherin binding to β-catenin also blocked binding of the androgen receptor LBD. We also demonstrated cross talk between the WNT and androgen receptor signaling pathways because excess androgen receptor could interfere with WNT signaling and excess TCF-4 inhibited the interaction of β-catenin and androgen receptor. Taken together, the data show that β-catenin can bind to the

  14. Serum and testicular testosterone and androgen binding protein profiles following subchronic treatment with carbendazim.

    PubMed

    Rehnberg, G L; Cooper, R L; Goldman, J M; Gray, L E; Hein, J F; McElroy, W K

    1989-10-01

    While the general toxicity of the benzimidazole pesticides for mammals is low, one of these compounds, carbendazim (MBC), causes degeneration of testicular tissue and decreases spermatogenic activity at doses well below the LD50 value. A study conducted by S. D. Carter, R. A. Hess, and J. W. Laskey (1987, Biol. Reprod. 37, 709-717) showed that treatment with 400 mg/kg/day MBC resulted in severe seminiferous tubular atrophy and infertility. Since spermatogenesis is an androgen-dependent process, we characterized the effects of MBC (0-400 mg/kg/day) on the endocrine function of the rat testes. Following subchronic (85 day) exposure, serum hormones (TSH, LH, FSH, and Prl) were measured as were androgen binding protein (ABP) and testosterone in testicular fluids (interstitial fluid and seminiferous tubule fluid). In addition, the functional capacity of the Leydig cell to secrete testosterone was assessed in vitro following an hCG challenge. Subchronic treatment with MBC at doses of 50-100 mg/kg/day had no effect on pituitary or testicular hormone concentrations: 200 mg/kg/day elevated the testosterone concentration in the seminiferous tubule fluid and the ABP concentration in both the interstitial fluid and the seminiferous tubule fluid without affecting serum testosterone or ABP concentrations. The 400 mg/kg/day dose resulted in increased concentration of both testosterone and ABP in the interstitial fluid and seminiferous tubule fluid and elevated serum ABP, with no change in serum testosterone. This endocrine profile is consistent with the testicular atrophy and "Sertoli cell-only" syndrome seen in these animals as reported by Gray et al. (1987, Toxicologist 7, 717). We conclude that seminiferous tubule fluid testosterone may be a result of two factors: (1) increased interstitial fluid testosterone concentrations and (2) decreased testosterone outflow from the testis to the general circulation. Also, increased ABP in the interstitial fluid may reflect a change in

  15. Characterization of α-Crystallin-Plasma Membrane Binding*

    PubMed Central

    Cobb, Brian A.; Petrash, J. Mark

    2010-01-01

    α-Crystallin, a large lenticular protein complex made up of two related subunits (αA- and αB-crystallin), is known to associate increasingly with fiber cell plasma membranes with age and/or the onset of cataract. To understand better the binding mechanism, we developed a sensitive membrane binding assay using lens plasma membranes and recombinant human αA- and αB-crystallins conjugated to a small fluorescent tag (Alexa350®). Both αA and αB homopolymer complexes, as well as a reconstituted 3:1 heteromeric complex, bind to lens membranes in a specific, saturable, and partially irreversible manner that is sensitive to both time and temperature. The amount of α-crystallin that binds to the membrane increases under acidic pH conditions and upon removal of exposed intrinsic membrane protein domains but is not affected at high ionic strength, suggesting that α-crystallin binds to the fiber cell plasma membranes mainly through hydrophobic interactions. The binding capacity and affinity for the reconstituted 3:1 heteromeric complex were measured to be 3.45 ± 0.11 ng/μg of membrane and 4.57 ± 0.50 × 10−4 μg−1 of membrane, respectively. The present membrane binding data support the hypothesis that the physical properties of a mixed α-crystallin complex may hold particular relevance for the function of α-crystallin within the lens. PMID:10692476

  16. Inhibition of renal membrane binding and nephrotoxicity of aminoglycosides

    SciTech Connect

    Williams, P.D.; Hottendorf, G.H.; Bennett, D.B.

    1986-06-01

    The initial event in the renal tubular reabsorption of nephrotoxic aminoglycosides involves binding to brush border membranes. This primary event was measured in renal brush border membrane vesicles prepared from rat renal cortex utilizing (3H)gentamicin. In order to gain structure-activity information regarding this interaction the effect of substances having chemical similarities to aminoglycosides (sugars, polyamines and amino acids) on gentamicin binding to brush border membranes was determined. Polyamino acids were found to possess the greatest inhibitory potency. In addition to polymers of cationic amino acids (lysine, ornithine, arginine and histidine), polymers of neutral (asparagine) and acidic (aspartic and glutamic acid) amino acids also exhibited inhibition of the membrane binding of gentamicin. Inasmuch as inhibition of renal membrane binding has the potential to decrease aminoglycoside nephrotoxicity, several polyamino acids that inhibited membrane binding were tested in vivo for potential protective activity vs. gentamicin- and amikacin-induced nephrotoxicity. Polyasparagine90 and polyaspartic acid100 inhibited gentamicin and amikacin nephrotoxicity completely when coadministered to rats with the aminoglycosides. Polylysine20 provided complete and partial inhibition of gentamicin and amikacin nephrotoxicity, respectively. Whereas in vivo distribution studies revealed that cortical levels of (3H)amikacin were elevated slightly by the coadministration of polyaspartic acid, brush border and basolateral membranes contained significantly lower levels of the aminoglycoside (46 and 41% inhibition, respectively). These results question the role of charge per se in the binding of aminoglycosides to renal membranes and further confirm the importance of membrane binding in the pathogenesis of aminoglycoside nephrotoxicity.

  17. A preliminary MTD-PLS study for androgen receptor binding of steroid compounds

    NASA Astrophysics Data System (ADS)

    Bora, Alina; Seclaman, E.; Kurunczi, L.; Funar-Timofei, Simona

    The relative binding affinities (RBA) of a series of 30 steroids for Human Androgen Receptor (AR) were used to initiate a MTD-PLS study. The 3D structures of all the compounds were obtained through geometry optimization in the framework of AM1 semiempirical quantum chemical method. The MTD hypermolecule (HM) was constructed, superposing these structures on the AR-bonded dihydrotestosterone (DHT) skeleton obtained from PDB (AR complex, ID 1I37). The parameters characterizing the HM vertices were collected using: AM1 charges, XlogP fragmental values, calculated fragmental polarizabilities (from refractivities), volumes, and H-bond parameters (Raevsky's thermodynamic originated scale). The resulted QSAR data matrix was submitted to PCA (Principal Component Analysis) and PLS (Projections in Latent Structures) procedure (SIMCA P 9.0); five compounds were selected as test set, and the remaining 25 molecules were used as training set. In the PLS procedure supplementary chemical information was introduced, i.e. the steric effect was always considered detrimental, and the hydrophobic and van der Waals interactions were imposed to be beneficial. The initial PLS model using the entire training set has the following characteristics: R2Y = 0.584, Q2 = 0.344. Based on distances to the model criterions (DMODX and DMODY), five compounds were eliminated and the obtained final model had the following characteristics: R2Y D 0.891, Q2 D 0.591. For this the external predictivity on the test set was unsatisfactory. A tentative explanation for these behaviors is the weak information content of the input QSAR matrix for the present series comparatively with other successful MTD-PLS modeling published elsewhere.

  18. In Situ Quantification of Protein Binding to the Plasma Membrane

    PubMed Central

    Smith, Elizabeth M.; Hennen, Jared; Chen, Yan; Mueller, Joachim D.

    2015-01-01

    This study presents a fluorescence-based assay that allows for direct measurement of protein binding to the plasma membrane inside living cells. An axial scan through the cell generates a fluorescence intensity profile that is analyzed to determine the membrane-bound and cytoplasmic concentrations of a peripheral membrane protein labeled by the enhanced green fluorescent protein (EGFP). The membrane binding curve is constructed by mapping those concentrations for a population of cells with a wide range of protein expression levels, and a fit of the binding curve determines the number of binding sites and the dissociation coefficient. We experimentally verified the technique, using myosin-1C-EGFP as a model system and fit its binding curve. Furthermore, we studied the protein-lipid interactions of the membrane binding domains from lactadherin and phospholipase C-δ1 to evaluate the feasibility of using competition binding experiments to identify specific lipid-protein interactions in living cells. Finally, we applied the technique to determine the lipid specificity, the number of binding sites, and the dissociation coefficient of membrane binding for the Gag matrix domain of human T-lymphotropic virus type 1, which provides insight into early assembly steps of the retrovirus. PMID:26039166

  19. Deletion of the steroid-binding domain of the human androgen receptor gene in one family with complete androgen insensitivity syndrome: Evidence for further genetic heterogeneity in this syndrome

    SciTech Connect

    Brown, T.R.; Lubahn, D.B.; Wilson, E.M.; Joseph, D.R.; French, F.S.; Migeon, C.J. )

    1988-11-01

    The cloning of a cDNA for the human androgen receptor gene has resulted in the availability for cDNA probes that span various parts of the gene, including the entire steroid-binding domain and part of the DNA-binding domain, as well as part of the 5' region of the gene. The radiolabeled probes were used to screen for androgen receptor mutations on Southern blots prepared by restriction endonuclease digestion of genomic DNA from human subjects with complete androgen insensitivity syndrome (AIS). In this investigation, the authors considered only patients presenting complete AIS and with the androgen receptor (-) form as the most probably subjects to show a gene deletion. One subject from each of six unrelated families with the receptor (-) form of complete AIS and 10 normal subjects were studied. In the 10 normal subjects and in 5 of the 6 patients, identical DNA restriction fragment patterns were observed with EcoRI and BamHI. Analysis of other members of this family confirmed the apparent gene deletion. The data provide direct proof that complete AIS in some families can result from a deletion of the androgen receptor structural gene. However, other families do not demonstrate such a deletion, suggesting that point mutations may also result in the receptor (-) form of complete AIS, adding further to the genetic heterogeneity of this syndrome.

  20. Binding contribution between synaptic vesicle membrane and plasma membrane proteins in neurons: an AFM study.

    PubMed

    Sritharan, K C; Quinn, A S; Taatjes, D J; Jena, B P

    1998-01-01

    The final step in the exocytotic process is the docking and fusion of membrane-bound secretory vesicles at the cell plasma membrane. This docking and fusion is brought about by several participating vesicle membrane, plasma membrane and soluble cytosolic proteins. A clear understanding of the interactions between these participating proteins giving rise to vesicle docking and fusion is essential. In this study, the binding force profiles between synaptic vesicle membrane and plasma membrane proteins have been examined for the first time using the atomic force microscope. Binding force contributions of a synaptic vesicle membrane protein VAMP1, and the plasma membrane proteins SNAP-25 and syntaxin, are also implicated from these studies. Our study suggests that these three proteins are the major, if not the only contributors to the interactive binding force that exist between the two membranes. PMID:10452835

  1. Polyester monomers lack ability to bind and activate both androgenic and estrogenic receptors as determined by In Vitro and In Silico methods

    PubMed Central

    Osimitz, Thomas G.; Welsh, William J.; Ai, Ni; Toole, Colleen

    2015-01-01

    The paper presents results from the screening of seven monomers used by Eastman Chemical to make various polymers. Ethylene glycol, diethylene glycol, polytetramethylene glycol, isophthalic acid, monosodium-5-sulfoisophthalic acid, 1,4-cyclohexanedicarboxylic acid, and dimethylcyclohexanedicarboxylate were screened for potential androgenicity or estrogenicity. The following studies were conducted: QSAR for binding to the AR and ER, in vitro Androgen Receptor Binding Assay, in vitro Estrogen Receptor Binding Assays (alpha and beta isoforms), in vitro Androgen Receptor Transactivation Assay in human cells, and in vitro Estrogen Receptor Transactivation Assay in human cells. None of the QSAR models predicted that any of the monomers possessed appreciable binding affinity for either AR or ER. Binding assays showed no evidence of interaction with either the AR or the alpha or beta ER receptors. Similarly, the AR and ER transactivation assays were negative. Moreover, six of the seven monomers have been subjected to 13-week and developmental toxicity studies in rats with no androgen- or estrogen-related effects being noted. Given the negative results of the in vitro screening assays (except PMG which demonstrated cytotoxicity) as well as available repeated dose and developmental and reproductive studies, the data suggest that none of the monomers tested exhibit androgenic or estrogenic hazards. PMID:25455886

  2. A crayfish insulin-like-binding protein: another piece in the androgenic gland insulin-like hormone puzzle is revealed.

    PubMed

    Rosen, Ohad; Weil, Simy; Manor, Rivka; Roth, Ziv; Khalaila, Isam; Sagi, Amir

    2013-08-01

    Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatics analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which according to three-dimensional modeling is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a "pulldown" methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23-38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development. PMID:23775079

  3. Dose-dependent effects of small-molecule antagonists on the genomic landscape of androgen receptor binding

    PubMed Central

    2012-01-01

    Background The androgen receptor plays a critical role throughout the progression of prostate cancer and is an important drug target for this disease. While chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-Seq) is becoming an essential tool for studying transcription and chromatin modification factors, it has rarely been employed in the context of drug discovery. Results Here we report changes in the genome-wide AR binding landscape due to dose-dependent inhibition by drug-like small molecules using ChIP-Seq. Integration of sequence analysis, transcriptome profiling, cell viability assays and xenograft tumor growth inhibition studies enabled us to establish a direct cistrome-activity relationship for two novel potent AR antagonists. By selectively occupying the strongest binding sites, AR signaling remains active even when androgen levels are low, as is characteristic of first-line androgen ablation therapy. Coupled cistrome and transcriptome profiling upon small molecule antagonism led to the identification of a core set of AR direct effector genes that are most likely to mediate the activities of targeted agents: unbiased pathway mapping revealed that AR is a key modulator of steroid metabolism by forming a tightly controlled feedback loop with other nuclear receptor family members and this oncogenic effect can be relieved by antagonist treatment. Furthermore, we found that AR also has an extensive role in negative gene regulation, with estrogen (related) receptor likely mediating its function as a transcriptional repressor. Conclusions Our study provides a global and dynamic view of AR’s regulatory program upon antagonism, which may serve as a molecular basis for deciphering and developing AR therapeutics. PMID:22849360

  4. Kinetic and Thermodynamic Characterization of Dihydrotestosterone-Induced Conformational Perturbations in Androgen Receptor Ligand-Binding Domain

    PubMed Central

    Jasuja, Ravi; Ulloor, Jagadish; Yengo, Christopher M.; Choong, Karen; Istomin, Andrei Y.; Livesay, Dennis R.; Jacobs, Donald J.; Swerdloff, Ronald S.; Mikšovská, Jaroslava; Larsen, Randy W.; Bhasin, Shalender

    2009-01-01

    Ligand-induced conformational perturbations in androgen receptor (AR) are important in coactivator recruitment and transactivation. However, molecular rearrangements in AR ligand-binding domain (AR-LBD) associated with agonist binding and their kinetic and thermodynamic parameters are poorly understood. We used steady-state second-derivative absorption and emission spectroscopy, pressure and temperature perturbations, and 4,4′-bis-anilinonaphthalene 8-sulfonate (bis-ANS) partitioning to determine the kinetics and thermodynamics of the conformational changes in AR-LBD after dihydrotestosterone (DHT) binding. In presence of DHT, the second-derivative absorption spectrum showed a red shift and a change in peak-to-peak distance. Emission intensity increased upon DHT binding, and center of spectral mass was blue shifted, denoting conformational changes resulting in more hydrophobic environment for tyrosines and tryptophans within a more compact DHT-bound receptor. In pressure perturbation calorimetry, DHT-induced energetic stabilization increased the Gibbs free energy of unfolding to 8.4 ± 1.3 kcal/mol from 3.5 ± 1.6 kcal/mol. Bis-ANS partitioning studies revealed that upon DHT binding, AR-LBD underwent biphasic rearrangement with a high activation energy (13.4 kcal/mol). An initial, molten globule-like burst phase (k ∼30 sec−1) with greater solvent accessibility was followed by rearrangement (k ∼0.01 sec−1), leading to a more compact conformation than apo-AR-LBD. Molecular simulations demonstrated unique sensitivity of tyrosine and tryptophan residues during pressure unfolding with rearrangement of residues in the coactivator recruitment surfaces distant from the ligand-binding pocket. In conclusion, DHT binding leads to energetic stabilization of AR-LBD domain and substantial rearrangement of residues distant from the ligand-binding pocket. DHT binding to AR-LBD involves biphasic receptor rearrangement including population of a molten globule

  5. Membrane adsorbers comprising grafted glycopolymers for targeted lectin binding

    PubMed Central

    Chenette, Heather C.S.; Husson, Scott M.

    2014-01-01

    This work details the design and testing of affinity membrane adsorbers for lectin purifications that incorporate glucose-containing glycopolymers. It is the selective interaction between the sugar residues of the glycopolymer and the complementary carbohydrate-binding domain of the lectin that provides the basis for the isolation and purification of lectins from complex biological media. The design approach used in these studies was to graft glycopolymer ‘tentacles’ from macroporous regenerated cellulose membranes by atom transfer radical polymerization. As shown in earlier studies, this design approach can be used to prepare high-productivity membrane adsorbers. The model lectin, concanavalin A (conA), was used to evaluate membrane performance in bind-and-elute purification, using a low molecular weight sugar for elution. The membrane capacity for binding conA was measured at equilibrium and under dynamic conditions using flow rates of 0.1 and 1.0 mL/min. The first Damkohler number was estimated to relate the adsorption rate to the convective mass transport rate through the membrane bed. It was used to assess whether adsorption kinetics or mass transport contributed the primary limitation to conA binding. Analyses indicate that this system is not limited by the accessibility of the binding sites, but by the inherent rate of adsorption of conA onto the glycopolymer. PMID:25866416

  6. Cation-binding Capacity of Membranes Isolated from Micrococcus lysodeikticus

    PubMed Central

    Cutinelli, C.; Galdiero, F.; Tufano, M. A.

    1969-01-01

    A study was made of H+, Na+, K+, Ca++, and Mg++ binding and ion-exchange properties of the plasma-mesosome membrane system isolated from Micrococcus lysodeikticus strain NCTC 2665. Titration curves were obtained on membranes prepared according to the method of M. R. J. Salton and further exposed to pH 4 for 4 hr (membranes-H). The dissociation coefficients and binding capacities were obtained by applying the mass law equation and the plot of G. Schatchard to the data. The membranes-H possess four kinds of dissociable groups with pK 4.96, 4.18, 3.60, and 3.09, respectively, and a total binding capacity of 0.65 meq/g (dry weight). Potentiometric titrations of cations in the presence and in the absence of membranes-H show that cations (Na+, K+, Ca++, and Mg++) are bound by the dissociated groups of the membrane. The fall in pH value for bivalent cations is greater than that for monovalent cations. Cations of the same valency produce equal diminutions on pH. Furthermore, ion-exchange tests carried out on membranes saturated with Mg++ or Na+ and suspended in a medium containing 45Ca show that the cations are reversibly bound. PMID:5344091

  7. Oxytocin binding by myoepithelial cell membranes from involuted mammary tissue.

    PubMed

    Ruberti, A; Olins, G M; Eakle, K A; Bremel, R D

    1983-04-29

    Oxytocin binding activity of myoepithelial cell membranes from mammary tissue was measured under a variety of different experimental conditions. Mammary tissue from non-lactating rats bound oxytocin with a Kd of 9.2 +/- 1.6 nM (+/- S.E.) and indicates that receptors are retained by the myoepithelial cells in a non-lactating state. Ovariectomy of non-lactating rats did not depress the binding activity of the membranes. Administration of the estrogenic compounds estradiol-17 beta and diethylstibestrol at doses which affect uterine weight and are known to increase uterine oxytocin binding did not influence the binding activity of the myoepithelial cells. This indicates that the oxytocin receptors of the mammary gland are not under the same endocrine control as the uterine receptors. PMID:6303330

  8. RECOMBINANT ANDROGEN RECEPTOR (AR) BINDING ACROSS VERTEBRATE SPECIES: COMPARISON OF BINDING OF ENVIRONMENTAL COMPOUNDS TO HUMAN, RAINBOW TROUT AND FATHEAD MINNOW AR.

    EPA Science Inventory

    In vitro screening assays designed to identify androgen mimics or antagonists typically use mammalian (rat, human) androgen receptors (AR). Although the amino acid sequences of receptors from nonmammalian vertebrates are not identical to the mammalian receptors, it is uncertain ...

  9. Discovery of novel membrane binding structures and functions

    PubMed Central

    Kufareva, Irina; Lenoir, Marc; Dancea, Felician; Sridhar, Pooja; Raush, Eugene; Bissig, Christin; Gruenberg, Jean; Abagyan, Ruben; Overduin, Michael

    2014-01-01

    The function of a protein is determined by its intrinsic activity in the context of its subcellular distribution. Membranes localize proteins within cellular compartments and govern their specific activities. Discovering such membrane-protein interactions is important for understanding biological mechanisms, and could uncover novel sites for therapeutic intervention. Here we present a method for detecting membrane interactive proteins and their exposed residues that insert into lipid bilayers. Although the development process involved analysis of how C1b, C2, ENTH, FYVE, Gla, pleckstrin homology (PH) and PX domains bind membranes, the resulting Membrane Optimal Docking Area (MODA) method yields predictions for a given protein of known three dimensional structures without referring to canonical membrane-targeting modules. This approach was tested on the Arf1 GTPase, ATF2 acetyltransferase, von Willebrand factor A3 domain and Neisseria gonorrhoeae MsrB protein, and further refined with membrane interactive and non-interactive FAPP1 and PKD1 pleckstrin homology domains, respectively. Furthermore we demonstrate how this tool can be used to discover unprecedented membrane binding functions as illustrated by the Bro1 domain of Alix, which was revealed to recognize lysobisphosphatidic acid (LBPA). Validation of novel membrane-protein interactions relies on other techniques such as nuclear magnetic resonance spectroscopy (NMR) which was used here to map the sites of micelle interaction. Together this indicates that genome-wide identification of known and novel membrane interactive proteins and sites is now feasible, and provides a new tool for functional annotation of the proteome. PMID:25394204

  10. Discovery of novel membrane binding structures and functions.

    PubMed

    Kufareva, Irina; Lenoir, Marc; Dancea, Felician; Sridhar, Pooja; Raush, Eugene; Bissig, Christin; Gruenberg, Jean; Abagyan, Ruben; Overduin, Michael

    2014-12-01

    The function of a protein is determined by its intrinsic activity in the context of its subcellular distribution. Membranes localize proteins within cellular compartments and govern their specific activities. Discovering such membrane-protein interactions is important for understanding biological mechanisms and could uncover novel sites for therapeutic intervention. We present a method for detecting membrane interactive proteins and their exposed residues that insert into lipid bilayers. Although the development process involved analysis of how C1b, C2, ENTH, FYVE, Gla, pleckstrin homology (PH), and PX domains bind membranes, the resulting membrane optimal docking area (MODA) method yields predictions for a given protein of known three-dimensional structures without referring to canonical membrane-targeting modules. This approach was tested on the Arf1 GTPase, ATF2 acetyltransferase, von Willebrand factor A3 domain, and Neisseria gonorrhoeae MsrB protein and further refined with membrane interactive and non-interactive FAPP1 and PKD1 pleckstrin homology domains, respectively. Furthermore we demonstrate how this tool can be used to discover unprecedented membrane binding functions as illustrated by the Bro1 domain of Alix, which was revealed to recognize lysobisphosphatidic acid (LBPA). Validation of novel membrane-protein interactions relies on other techniques such as nuclear magnetic resonance spectroscopy (NMR), which was used here to map the sites of micelle interaction. Together this indicates that genome-wide identification of known and novel membrane interactive proteins and sites is now feasible and provides a new tool for functional annotation of the proteome. PMID:25394204

  11. Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes.

    PubMed

    De Gerónimo, Eduardo; Hagan, Robert M; Wilton, David C; Córsico, Betina

    2010-09-01

    Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates. PMID:20541621

  12. Stereospecific binding of 3H-phencyclidine in brain membranes.

    PubMed

    Hampton, R Y; Medzihradsky, F; Woods, J H; Dahlstrom, P J

    1982-06-21

    Phencyclidine (PCP) displaceable binding of 3H-PCP to glass-fiber filters was eliminated and total binding markedly reduced by initial treatment of the discs with 0.05% polyethyleneimine. Assessed with treated filters, unlabeled PCP displaced 3H-PCP in both rat and pigeon brain membranes with an EC50 of 1 microM. Of similar high inhibitory potency were dextrorphan, levorphanol, SKF 10047 and ketamine, while morphine, naloxone and etorphine had EC50 values higher then 1 mM. Using the dissociative anesthetic dexoxadrol and its inactive isomer levoxadrol as displacing agents, stereospecific binding of 3H-PCP was obtained in rat and pigeon brain membranes. The markedly higher potency of dexoxadrol, relative to levoxadrol, in displacing bound 3H-PCP is compatible with behavioral data for these enantiomers. However, they were equipotent in displacing 3H-PCP bound to glass-fiber filters in the absence of tissue. Heat denaturation, but not freezing, abolished stereospecific binding of 3H-PCP, which was also absent in rat liver membranes. The stereospecific binding component in brain displayed biphasic saturability at 60-70 nM and 300-400 nM, respectively. PMID:7109842

  13. F-actin binding regions on the androgen receptor and huntingtin increase aggregation and alter aggregate characteristics.

    PubMed

    Angeli, Suzanne; Shao, Jieya; Diamond, Marc I

    2010-01-01

    Protein aggregation is associated with neurodegeneration. Polyglutamine expansion diseases such as spinobulbar muscular atrophy and Huntington disease feature proteins that are destabilized by an expanded polyglutamine tract in their N-termini. It has previously been reported that intracellular aggregation of these target proteins, the androgen receptor (AR) and huntingtin (Htt), is modulated by actin-regulatory pathways. Sequences that flank the polyglutamine tract of AR and Htt might influence protein aggregation and toxicity through protein-protein interactions, but this has not been studied in detail. Here we have evaluated an N-terminal 127 amino acid fragment of AR and Htt exon 1. The first 50 amino acids of ARN127 and the first 14 amino acids of Htt exon 1 mediate binding to filamentous actin in vitro. Deletion of these actin-binding regions renders the polyglutamine-expanded forms of ARN127 and Htt exon 1 less aggregation-prone, and increases the SDS-solubility of aggregates that do form. These regions thus appear to alter the aggregation frequency and type of polyglutamine-induced aggregation. These findings highlight the importance of flanking sequences in determining the propensity of unstable proteins to misfold. PMID:20140226

  14. Androgen regulation of corticotropin-releasing hormone receptor 2 (CRHR2) mRNA expression and receptor binding in the rat brain

    PubMed Central

    Weiser, Michael J.; Goel, Nirupa; Sandau, Ursula S.; Bale, Tracy L.; Handa, Robert J.

    2008-01-01

    Stress-induced affective disorders, such as depression and anxiety, are more prevalent in females than in males. The reduced vulnerability to these disorders in males may be due to the presence of androgens, which are known to dampen the stress response and reduce anxiety-like behaviors. However, a neurobiological mechanism for this sex difference has yet to be elucidated. Corticotropin-releasing hormone receptor 2 (CRHR2) has been implicated in regulating anxiety-type behaviors and is expressed in stress-responsive brain regions that also contain androgen receptors (AR). We hypothesized that androgen may exert its effects through actions on CRHR2 and we therefore examined the regulation of CRHR2 mRNA and receptor binding in the male rat forebrain following androgen administration. Young adult male Sprague/Dawley rats were gonadectomized (GDX) and treated with the non-aromatizable androgen, dihydrotestosterone propionate (DHTP) using hormone filled Silastic capsules. Control animals received empty capsules. Using quantitative real time RT-PCR, CRHR2 mRNA levels were determined in block dissected brain regions. DHTP treatment significantly increased CRHR2 mRNA expression in the hippocampus, hypothalamus, and lateral septum (p < 0.01) when compared to vehicle-treated controls. A similar trend was observed in amygdala (p = 0.05). Furthermore, in vitro autoradiography revealed significantly higher CRHR2 binding in the lateral septum in androgen-treated males, with the highest difference observed in the ventral lateral region. Regulation of CRHR2 mRNA by AR was also examined using an in vitro approach. Hippocampal neurons, which contain high levels of AR, were harvested from E17–18 rat fetuses, and maintained in primary culture for 14 days. Neurons were then treated with dihydrotestosterone (DHT; 1 nM), DHT plus flutamide (an androgen receptor antagonist), or vehicle for 48 hours. CRHR2 mRNA levels were measured using quantitative real time RT-PCR. Consistent with in

  15. Polyamine Binding to Plasma Membrane Vesicles Isolated from Zucchini Hypocotyls.

    PubMed Central

    Tassoni, A.; Antognoni, F.; Bagni, N.

    1996-01-01

    The general features of [14C]spermidine binding to plasmalemma vesicles isolated from zucchini (Cucurbita pepo L.) etiolated hypocotyls are reported in the present paper. The specific interaction of the polyamine with the plasma membranes was reversible and thermolabile, since it decreased by about 50% in the assay performed at 40[deg]C compared to that carried out on ice. On the contrary, nonspecific binding was unaffected by temperature. Specific spermidine binding showed a pH dependence with a maximum at pH 8.0 and it reached saturation between 0.75 and 1 mM external spermidine concentration. The value of the dissociation constant calculated from Scatchard analysis was 4.4 x 10-5 M. Specific spermidine interaction appeared to be sensitive to detergents and was markedly reduced by the presence of divalent cations, such as Mg2+ and Ca2+, whereas it was stimulated by monovalent cations. Polyamine binding sites were highly sensitive to pronase treatment. Competition experiments, performed using a series of compounds structurally related to spermidine, may provide some indication of the characteristics of spermidine binding sites. The results presented here suggest that specific spermidine binding occurs mainly with the protein component of the plasma membrane. PMID:12226221

  16. Membrane-Binding Mechanism of Clostridium perfringens Alpha-Toxin

    PubMed Central

    Oda, Masataka; Terao, Yutaka; Sakurai, Jun; Nagahama, Masahiro

    2015-01-01

    Clostridium perfringens alpha-toxin is a key mediator of gas gangrene, which is a life-threatening infection that manifests as fever, pain, edema, myonecrosis, and gas production. Alpha-toxin possesses phospholipase C and sphingomyelinase activities. The toxin is composed of an N-terminal domain (1–250 aa, N-domain), which is the catalytic site, and a C-terminal domain (251–370 aa, C-domain), which is the membrane-binding site. Immunization of mice with the C-domain of alpha-toxin prevents the gas gangrene caused by C. perfringens, whereas immunization with the N-domain has no effect. The central loop domain (55–93 aa), especially H….SW84Y85….G, plays an important role in the interaction with ganglioside GM1a. The toxin binds to lipid rafts in the presence of a GM1a/TrkA complex, and metabolites from phosphatidylcholine to diacylglycerol through the enzymatic activity of alpha-toxin itself. These membrane dynamics leads to the activation of endogenous PLCγ-1 via TrkA. In addition, treatment with alpha-toxin leads to the formation of diacylglycerol at membrane rafts in ganglioside-deficient DonQ cells; this in turn triggers endocytosis and cell death. This article summarizes the current the membrane-binding mechanism of alpha-toxin in detail. PMID:26633512

  17. Homology-modeled ligand-binding domains of medaka estrogen receptors and androgen receptors: A model system for the study of reproduction

    SciTech Connect

    Cui Jianzhou Shen Xueyan; Yan Zuowei; Zhao Haobin; Nagahama, Yoshitaka

    2009-02-27

    Estrogen and androgen and their receptors play critical roles in physiological processes such as sexual differentiation and development. Using the available structural models for the human estrogen receptors alpha and beta and androgen receptor as templates, we designed in silico agonist and antagonist models of medaka estrogen receptor (meER) alpha, beta-1, and beta-2, and androgen receptor (meAR) alpha and beta. Using these models, we studied (1) the structural relationship between the ligand-binding domains (LBDs) of ERs and ARs of human and medaka, and (2) whether medaka ER and AR can be potential models for studying the ligand-binding activities of various agonists and antagonists of these receptors by docking analysis. A high level of conservation was observed between the sequences of the ligand-binding domains of meER{alpha} and huER{alpha}, meER{beta}1 and huER{beta}, meER{beta}2, and huER{beta} with 62.8%, 66.4%, and 65.1% identity, respectively. The sequence conservation between meAR{alpha} and huAR, meAR{beta}, and huAR was found with 70.1% and 61.0% of identity, respectively. Thirty-three selected endocrine disrupting chemicals (EDCs), including both agonists and antagonists, were docked into the LBD of ER and AR, and the corresponding docking score for medaka models and human templates were calculated. In order to confirm the conservation of the overall geometry and the binding pocket, the backbone root mean square deviation (RMSD) for C{alpha} atoms was derived from the structure superposition of all 10 medaka homology models to the six human templates. Our results suggested conformational conservation between the ERs and ARs of medaka and human, Thus, medaka could be highly useful as a model system for studies involving estrogen and androgen interaction with their receptors.

  18. Structural Diversity of Ligand-Binding Androgen Receptors Revealed by Microsecond Long Molecular Dynamics Simulations and Enhanced Sampling.

    PubMed

    Duan, Mojie; Liu, Na; Zhou, Wenfang; Li, Dan; Yang, Minghui; Hou, Tingjun

    2016-09-13

    Androgen receptor (AR) plays important roles in the development of prostate cancer (PCa). The antagonistic drugs, which suppress the activity of AR, are widely used in the treatment of PCa. However, the molecular mechanism of antagonism about how ligands affect the structures of AR remains elusive. To better understand the conformational variability of ARs bound with agonists or antagonists, we performed long time unbiased molecular dynamics (MD) simulations and enhanced sampling simulations for the ligand binding domain of AR (AR-LBD) in complex with various ligands. Based on the simulation results, we proposed an allosteric pathway linking ligands and helix 12 (H12) of AR-LBD, which involves the interactions among the ligands and the residues W741, H874, and I899. The interaction pathway provides an atomistic explanation of how ligands affect the structure of AR-LBD. A repositioning of H12 was observed, but it is facilitated by the C-terminal of H12, instead of by the loop between helix 11 (H11) and H12. The bias-exchange metadynamics simulations further demonstrated the above observations. More importantly, the free energy profiles constructed by the enhanced sampling simulations revealed the transition process between the antagonistic form and agonistic form of AR-LBD. Our results would be helpful for the design of more efficient antagonists of AR to combat PCa. PMID:27560203

  19. Antiviral activity of squalamine: Role of electrostatic membrane binding

    NASA Astrophysics Data System (ADS)

    Beckerman, Bernard; Qu, Wei; Mishra, Abhijit; Zasloff, Michael; Wong, Gerard; Luijten, Erik

    2012-02-01

    Recent workootnotetextM. Zasloff et al., Proc. Nat. Acad. Sci. (USA) 108, 15978 (2011). has demonstrated that squalamine, a molecule found in the liver of sharks, exhibits broad-spectrum antiviral properties. It has been proposed that this activity results from the charge-density matching of squalamine and phospholipid membranes, causing squalamine to bind to membranes and displace proteins such as Rac1 that are crucial for the viral replication cycle. Here we investigate this hypothesis by numerical simulation of a coarse-grained model for the competition between Rac1 and squalamine in binding affinity to a flat lipid bilayer. We perform free-energy calculations to test the ability of squalamine to condense stacked bilayer systems and thereby displace bulkier Rac1 molecules. We directly compare our findings to small-angle x-ray scattering results for the same setup.

  20. Two nucleotide binding sites modulate ( sup 3 H) glyburide binding to rat cortex membranes

    SciTech Connect

    Johnson, D.E.; Gopalakrishnan, M.; Triggle, D.J.; Janis, R.A. State Univ. of New York, Buffalo )

    1991-03-11

    The effects of nucleotides on the binding of the ATP-dependent K{sup +}-channel antagonist ({sup 3}H)glyburide (GLB) to rat cortex membranes were examined. Nucleotide triphosphates (NTPs) and nucleotide diphosphate (NDPs) inhibited the binding of GLB. This effect was dependent on the presence of dithiothreitol (DTT). Inhibition of binding by NTPs, with the exception of ATP{gamma}S, was dependent on the presence of Mg{sup 2+}. GLB binding showed a biphasic response to ADP: up to 3 mM, ADP inhibited binding, and above this concentration GLB binding increased rapidly, and was restored to normal levels by 10 mM ADP. In the presence of Mg{sup 2+}, ADP did not stimulate binding. Saturation analysis in the presence of Mg{sup 2+} and increasing concentrations of ADP showed that ADP results primarily in a change of the B{sub max} for GLB binding. The differential effects of NTPS and NDPs indicate that two nucleotide binding sites regulate GLB binding.

  1. Single Molecule Kinetics of ENTH Binding to Lipid Membranes

    SciTech Connect

    Rozovsky, Sharon; Forstner, Martin B.; Sondermann, Holger; Groves, Jay T.

    2012-04-03

    Transient recruitment of proteins to membranes is a fundamental mechanism by which the cell exerts spatial and temporal control over proteins’ localization and interactions. Thus, the specificity and the kinetics of peripheral proteins’ membrane residence are an attribute of their function. In this article, we describe the membrane interactions of the interfacial epsin N-terminal homology (ENTH) domain with its target lipid phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2). The direct visualization and quantification of interactions of single ENTH molecules with supported lipid bilayers is achieved using total internal reflection fluorescence microscopy (TIRFM) with a time resolution of 13 ms. This enables the recording of the kinetic behavior of ENTH interacting with membranes with physiologically relevant concentrations of PtdIns(4,5)P2 despite the low effective binding affinity. Subsequent single fluorophore tracking permits us to build up distributions of residence times and to measure ENTH dissociation rates as a function of membrane composition. In addition, due to the high time resolution, we are able to resolve details of the motion of ENTH associated with a simple, homogeneous membrane. In this case ENTH’s diffusive transport appears to be the result of at least three different diffusion processes.

  2. Modulation of membrane fusion by calcium-binding proteins.

    PubMed Central

    Hong, K; Düzgüneş, N; Papahadjopoulos, D

    1982-01-01

    The effects of several Ca2+-binding proteins (calmodulin, prothrombin, and synexin) on the kinetics of Ca2+-induced membrane fusion were examined. Membrane fusion was assayed by following the mixing of aqueous contents of phospholipid vesicles. Calmodulin inhibited slightly the fusion of phospholipid vesicles. Bovine prothrombin and its proteolytic fragment 1 had a strong inhibitory effect on fusion. Depending on the phospholipid composition, synexin could either facilitate or inhibit Ca2+-induced fusion of vesicles. The effects of synexin were Ca2+ specific. 10 microM Ca2+ was sufficient to induce fusion of vesicles composed of phosphatidic acid/phosphatidylethanolamine (1:3) in the presence of synexin and 1 mM Mg2+. We propose that synexin may be involved in intracellular membrane fusion events mediated by Ca2+, such as exocytosis, and discuss possible mechanisms facilitating fusion. PMID:6459804

  3. Membrane-Binding and Enzymatic Properties of RPE65

    PubMed Central

    Kiser, Philip D.; Palczewski, Krzysztof

    2010-01-01

    Regeneration of visual pigments is essential for sustained visual function. Although the requirement for non-photochemical regeneration of the visual chromophore, 11-cis-retinal, was recognized early on, it was only recently that the trans to cis retinoid isomerase activity required for this process was assigned to a specific protein, a microsomal membrane enzyme called RPE65. In this review, we outline progress that has been made in the functional characterization of RPE65. We then discuss general concepts related to protein-membrane interactions and the mechanism of the retinoid isomerization reaction and describe some of the important biochemical and structural features of RPE65 with respect to its membrane-binding and enzymatic properties. PMID:20304090

  4. Thymocyte plasma membrane: the location of specific glucocorticoid binding sites

    SciTech Connect

    Sergeev, P.V.; Kalinin, G.V.; Dukhanin, A.S.

    1987-01-01

    In modern molecular endocrinology it is now possible to determine the localization of receptors for biologically active substances with the aid of ligands, with high affinity for the receptor, immobilized on polymers. The purpose of this paper is to study the ability of hydrocortisone (HC), immobilized on polyvinylpyrrolidone (PVP-HC), to reduce binding of tritium-HC by thymocytes of adrenalectomized rats. It is determined that specific binding sites for HC on rat thymocytes are also accessible for PVP-HC, which, due to the fact that this immobilized version of HC does not penetrate into the cell, leads to the conclusion that the binding sites for HC itself are located in the plasma membrane.

  5. Cytoskeletal protein binding kinetics at planar phospholipid membranes.

    PubMed Central

    Mc Kiernan, A E; MacDonald, R I; MacDonald, R C; Axelrod, D

    1997-01-01

    It has been hypothesized that nonspecific reversible binding of cytoskeletal proteins to lipids in cells may guide their binding to integral membrane anchor proteins. In a model system, we measured desorption rates k(off) (off-rates) of the erythrocyte cytoskeletal proteins spectrin and protein 4.1 labeled with carboxyfluorescein (CF), at two different compositions of planar phospholipid membranes (supported on glass), using the total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP) technique. The lipid membranes consisted of either pure phosphatidylcholine (PC) or a 3:1 mixture of PC with phosphatidylserine (PS). In general, the off-rates were not single exponentials and were fit to a combination of fast, slow, and irreversible fractions, reported both separately and as a weighted average. By a variation of TIR/FRAP, we also measured equilibrium affinities (the ratio of surface-bound to bulk protein concentration) and thereby calculated on-rates, k(on). The average off-rate of CF-4.1 from PC/PS (approximately 0.008/s) is much slower than that from pure PC (approximately 1.7/s). Despite the consequent increase in equilibrium affinity at PC/PS, the on-rate at PC/PS is also substantially decreased (by a factor of 40) relative to that at pure PC. The simultaneous presence of (unlabeled) spectrin tends to substantially decrease the on-rate (and the affinity) of CF-4.1 at both membrane types. Similar experiments for CF-spectrin alone showed much less sensitivity to membrane type and generally faster off-rates than those exhibited by CF-4.1. However, when mixed with (unlabeled) 4.1, both the on-rate and off-rate of CF-spectrin decreased drastically at PC/PS (but not PC), leading to a somewhat increased affinity. Clearly, changes in affinity often involve countervailing changes in both on-rates and off-rates. In many of these studies, the effect of varying ionic strength and bulk concentrations was examined; it appears that the binding is an

  6. Characterization of PROPPIN-Phosphoinositide Binding and Role of Loop 6CD in PROPPIN-Membrane Binding

    PubMed Central

    Busse, Ricarda A.; Scacioc, Andreea; Krick, Roswitha; Pérez-Lara, Ángel; Thumm, Michael; Kühnel, Karin

    2015-01-01

    PROPPINs (β-propellers that bind polyphosphoinositides) are a family of PtdIns3P- and PtdIns(3,5)P2-binding proteins that play an important role in autophagy. We analyzed PROPPIN-membrane binding through isothermal titration calorimetry (ITC), stopped-flow measurements, mutagenesis studies, and molecular dynamics (MD) simulations. ITC measurements showed that the yeast PROPPIN family members Atg18, Atg21, and Hsv2 bind PtdIns3P and PtdIns(3,5)P2 with high affinities in the nanomolar to low-micromolar range and have two phosphoinositide (PIP)-binding sites. Single PIP-binding site mutants have a 15- to 30-fold reduced affinity, which explains the requirement of two PIP-binding sites in PROPPINs. Hsv2 bound small unilamellar vesicles with a higher affinity than it bound large unilamellar vesicles in stopped-flow measurements. Thus, we conclude that PROPPIN membrane binding is curvature dependent. MD simulations revealed that loop 6CD is an anchor for membrane binding, as it is the region of the protein that inserts most deeply into the lipid bilayer. Mutagenesis studies showed that both hydrophobic and electrostatic interactions are required for membrane insertion of loop 6CD. We propose a model for PROPPIN-membrane binding in which PROPPINs are initially targeted to membranes through nonspecific electrostatic interactions and are then retained at the membrane through PIP binding. PMID:25954880

  7. pMD-Membrane: A Method for Ligand Binding Site Identification in Membrane-Bound Proteins

    PubMed Central

    Gorfe, Alemayehu A.

    2015-01-01

    Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation. PMID:26506102

  8. Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes

    SciTech Connect

    Beschiaschvili, G.; Seelig, J. )

    1990-01-09

    The binding of bee venom melittin to negatively charged unilamellar vesicles and planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was studied with circular dichroism and deuterium NMR spectroscopy. The melittin binding isotherm was measured for small unilamellar vesicles containing 10 or 20 mol % POPG. Due to electrostatic attraction, binding of the positively charged melittin was much enhanced as compared to the binding to neutral lipid vesicles. However, after correction for electrostatic effects by means of the Gouy-Chapman theory, all melittin binding isotherms could be described by a partition Kp = (4.5 +/- 0.6) x 10(4) M-1. It was estimated that about 50% of the total melittin surface was embedded in a hydrophobic environment. The melittin partition constant for small unilamellar vesicles was by a factor of 20 larger than that of planar bilayers and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium NMR studies were performed with coarse lipid dispersions. Binding of melittin to POPC/POPG (80/20 mol/mol) membranes caused systematic changes in the conformation of the phosphocholine and phosphoglycerol head groups which were ascribed to the influence of electrostatic charge on the choline dipole. While the negative charge of phosphatidylglycerol moved the N+ end of the choline -P-N+ dipole toward the bilayer interior, the binding of melittin reversed this effect and rotated the N+ end toward the aqueous phase. No specific melittin-POPG complexes could be detected. The phosphoglycerol head group was less affected by melittin binding than its choline counterpart.

  9. Synaptotagmin-1 binds to PIP2-containing membrane but not to SNAREs at physiological ionic strength

    PubMed Central

    Park, Yongsoo; Seo, Jong Bae; Fraind, Alicia; Pérez-Lara, Angel; Yavuz, Halenur; Han, Kyungreem; Jung, Seung-Ryoung; Kattan, Iman; Walla, Peter Jomo; Choi, MooYoung; Cafiso, David S.; Koh, Duk-Su; Jahn, Reinhard

    2015-01-01

    Ca2+-sensor synaptotagmin-1 is thought to trigger membrane fusion by binding to acidic membrane lipids and SNARE proteins. Previous work has shown that binding is mediated by electrostatic interactions that are sensitive to the ionic environment. However, the influence of divalent or polyvalent ions, at physiological concentrations, on synaptotagmin binding to membranes or SNAREs has not been explored. Here we show that binding of rat synaptotagmin-1 to membranes containing PIP2 is regulated by charge shielding caused by the presence of divalent cations. Surprisingly, polyvalent ions such as ATP and Mg2+ completely abrogate synaptotagmin-1 binding to SNAREs regardless of whether Ca2+ is present or not. Altogether, our data suggest that at physiological ion concentrations Ca2+-dependent synaptotagmin-1 binding is confined to PIP2-containing membrane patches in the plasma membrane, suggesting that membrane interaction of synaptotagmin-1 rather than SNARE binding triggers exocytosis of vesicles. PMID:26389740

  10. A Model Membrane Protein for Binding Volatile Anesthetics

    PubMed Central

    Ye, Shixin; Strzalka, Joseph; Churbanova, Inna Y.; Zheng, Songyan; Johansson, Jonas S.; Blasie, J. Kent

    2004-01-01

    Earlier work demonstrated that a water-soluble four-helix bundle protein designed with a cavity in its nonpolar core is capable of binding the volatile anesthetic halothane with near-physiological affinity (0.7 mM Kd). To create a more relevant, model membrane protein receptor for studying the physicochemical specificity of anesthetic binding, we have synthesized a new protein that builds on the anesthetic-binding, hydrophilic four-helix bundle and incorporates a hydrophobic domain capable of ion-channel activity, resulting in an amphiphilic four-helix bundle that forms stable monolayers at the air/water interface. The affinity of the cavity within the core of the bundle for volatile anesthetic binding is decreased by a factor of 4–3.1 mM Kd as compared to its water-soluble counterpart. Nevertheless, the absence of the cavity within the otherwise identical amphiphilic peptide significantly decreases its affinity for halothane similar to its water-soluble counterpart. Specular x-ray reflectivity shows that the amphiphilic protein orients vectorially in Langmuir monolayers at higher surface pressure with its long axis perpendicular to the interface, and that it possesses a length consistent with its design. This provides a successful starting template for probing the nature of the anesthetic-peptide interaction, as well as a potential model system in structure/function correlation for understanding the anesthetic binding mechanism. PMID:15465862

  11. Ocelot and oncilla spermatozoa can bind hen egg perivitelline membranes.

    PubMed

    de Araujo, Gediendson Ribeiro; de Paula, Tarcizio Antônio Rego; Deco-Souza, Thyara de; Garay, Rafael de Morais; Letícia Bergo, C F; Csermak-Júnior, Antônio Carlos; da Silva, Leanes Cruz; Alves, Saullo Vinícius Pereira

    2015-12-01

    We evaluated the capacity of ocelot and oncilla spermatozoa to bind to the perivitelline membranes (PVMs) of hen eggs in a sperm binding assay (S-PVM). In addition, a device that improves the standardization of the assay was developed. The number of sperm bound to the PVM in fresh (T1) and frozen-thawed (T2) semen from both species was compared to the sperm quality observed in routine tests. The PVM was stretched on a circular silicone device to create a standardized area for analysis. In both treatments and for both species, the spermatozoa were able to bind to the PVM, indicating that PVM may be used for a sperm binding assay in ocelot and oncilla. The S-PVM assay did not differ in fresh and frozen-thawed ocelot sperm (p>0.05). However, fewer oncilla sperm (p<0.05) were bound to the PVM in T2, indicating that the proposed test may be able to detect injuries that compromise sperm binding abilities. The device maintained the PVM stretched during the processing and defined the evaluation area. PMID:26526118

  12. Characterization of auxin-binding proteins from zucchini plasma membrane

    NASA Technical Reports Server (NTRS)

    Hicks, G. R.; Rice, M. S.; Lomax, T. L.

    1993-01-01

    We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may

  13. Carotenoid binding to proteins: Modeling pigment transport to lipid membranes.

    PubMed

    Reszczynska, Emilia; Welc, Renata; Grudzinski, Wojciech; Trebacz, Kazimierz; Gruszecki, Wieslaw I

    2015-10-15

    Carotenoid pigments play numerous important physiological functions in human organism. Very special is a role of lutein and zeaxanthin in the retina of an eye and in particular in its central part, the macula lutea. In the retina, carotenoids can be directly present in the lipid phase of the membranes or remain bound to the protein-pigment complexes. In this work we address a problem of binding of carotenoids to proteins and possible role of such structures in pigment transport to lipid membranes. Interaction of three carotenoids, beta-carotene, lutein and zeaxanthin with two proteins: bovine serum albumin and glutathione S-transferase (GST) was investigated with application of molecular spectroscopy techniques: UV-Vis absorption, circular dichroism and Fourier transform infrared spectroscopy (FTIR). Interaction of pigment-protein complexes with model lipid bilayers formed with egg yolk phosphatidylcholine was investigated with application of FTIR, Raman imaging of liposomes and electrophysiological technique, in the planar lipid bilayer models. The results show that in all the cases of protein and pigment studied, carotenoids bind to protein and that the complexes formed can interact with membranes. This means that protein-carotenoid complexes are capable of playing physiological role in pigment transport to biomembranes. PMID:26361975

  14. Glucagon-like peptide-1 binding to rat hepatic membranes.

    PubMed

    Villanueva-Peñacarrillo, M L; Delgado, E; Trapote, M A; Alcántara, A; Clemente, F; Luque, M A; Perea, A; Valverde, I

    1995-07-01

    We have found [125I]glucagon-like peptide (GLP)-1(7-36)amide specific binding activity in rat liver and isolated hepatocyte plasma membranes, with an M(r) of approximately 63,000, estimated by cross-linking and SDS-PAGE. The specific binding was time- and membrane protein concentration-dependent, and equally displaced by unlabelled GLP-1(7-36)amide and by GLP-1(1-36)amide, achieving its ID50 at 3 x 10(-9) M of the peptides. GLP-1(7-36)amide did not modify the basal or the glucagon (10(-8) M)-stimulated adenylate cyclase in the hepatocyte plasma membranes. These data, together with our previous findings of a potent glycogenic effect of GLP-1(7-36)amide in isolated rat hepatocytes, led us to postulate that the insulin-like effects of this peptide on glucose liver metabolism could be mediated by a type of receptor probably different from that described for GLP-1 in pancreatic B-cells or, alternatively, by the same receptor which, in this tissue as well as in muscle, uses a different transduction system. PMID:7561616

  15. Regulation of expression of Na+,K+-ATPase in androgen-dependent and androgen-independent prostate cancer

    PubMed Central

    Blok, L J; Chang, G T G; Steenbeek-Slotboom, M; Weerden, W M van; Swarts, H G P; Pont, J J H H M De; Steenbrugge, G J van; Brinkmann, A O

    1999-01-01

    The β1-subunit of Na+,K+-ATPase was isolated and identified as an androgen down-regulated gene. Expression was observed at high levels in androgen-independent as compared to androgen-dependent (responsive) human prostate cancer cell lines and xenografts when grown in the presence of androgens. Down-regulation of the β1-subunit was initiated at concentrations between 0.01 nM and 0.03 nM of the synthetic androgen R1881 after relatively long incubation times (> 24 h). Using polyclonal antibodies, the concentration of β1-subunit protein, but not of the α1-subunit protein, was markedly reduced in androgen-dependent human prostate cancer cells (LNCaP-FGC) cultured in the presence of androgens. In line with these observations it was found that the protein expression of total Na+,K+-ATPase in the membrane (measured by 3H-ouabain binding) was also markedly decreased. The main function of Na+,K+-ATPase is to maintain sodium and potassium homeostasis in animal cells. The resulting electrochemical gradient is facilitative for transport of several compounds over the cell membrane (for example cisplatin, a chemotherapeutic agent experimentally used in the treatment of hormone-refractory prostate cancer). Here we observed that a ouabain-induced decrease of Na+,K+-ATPase activity in LNCaP-FGC cells results in reduced sensitivity of these cells to cisplatin-treatment. Surprisingly, androgen-induced decrease of Na+,K+-ATPase expression, did not result in significant protection against the chemotherapeutic agent. © 1999 Cancer Research Campaign PMID:10487609

  16. Ciprofloxacin encapsulation into giant unilamellar vesicles: membrane binding and release.

    PubMed

    Kaszás, Nóra; Bozó, Tamás; Budai, Marianna; Gróf, Pál

    2013-02-01

    This study aimed at investigating some respects of binding and interaction between water-soluble drugs and liposomal carrier systems depending on their size and lamellarity. As model substance, ciprofloxacin hydrochloride (CPFX) was incorporated into giant unilamellar vesicles (GUVs) to study their CPFX encapsulation/binding capacity. To characterize molecular interactions of various CPFX microspecies with lipid bilayer, zeta potential and electron paramagnetic resonance (EPR) spectroscopy measurements were performed. The increase of the zeta potential at pH 5.4 but no change at pH 7.2 was interpreted in terms of the CPFX microspecies' distribution at the two pH values. EPR observations showed an increased fluidity because of CPFX binding to GUVs. We worked out and applied a three-compartment dialysis model to separately determine the rate of drug diffusion through the liposomal membrane. Equilibrium dialysis showed (a) different permeation of CPFX through the membranes of GUVs and multilamellar vesicles (MLVs), with characteristic half-lives of 54.4 and 18.1 h, respectively; and (b) increased retention of CPFX in case of GUVs with released amounts of 70% compared with about 97% in case of MLVs. Our results may provide further details for efficient design of liposomal formulations incorporating water-soluble drugs. PMID:23233199

  17. Binding of fluorescent lanthanides to rat liver mitochondrial membranes and calcium ion-binding proteins.

    PubMed

    Mikkelsen, R B; Wallach, D F

    1976-05-21

    (1) Tb3+ binding to mitochondrial membranes can be monitored by enhanced ion fluorescence at 545 nm with excitation at 285 nm. At low protein concentrations (less than 30 mug/ml) no inner filter effects are observed. (2) This binding is localized at the external surface of the inner membrane and is unaffected by inhibitors of respiration or oxidative phosphorylation. (3) A soluble Ca2+ binding protein isolated according to Lehninger, A.L. ((1971) Biochem. Biophys. Res. Commun. 42, 312-317) also binds Tb3+ with enhanced ion fluorescence upon excitation at 285 nm. The excitation spectrum of the isolated protein and of the intact mitochondria are indicative of an aromatic amino acid at the cation binding site. (4) Further characterization of the Tb3+-protein interaction revealed that there is more than one binding site per protein molecule and that these sites are clustered (less than 20 A). Neuraminidase treatment or organic solvent extraction of the protein did not affect fluorescent Tb3+ binding. (5) pH dependency studies of Tb3+ binding to the isolated protein or intact mitochondria demonstrated the importance of an ionizable group of pK greater than 6. At pH less than 7.5 the amount of Tb3+ bound to the isolated protein decreased with increase in pH as monitored by Tb3+ fluorescence. With intact mitochondria the opposite occurred with a large increase in Tb3+ fluorescence at higher pH. This increase was not observed when the mitochondria were preincubated with antimycin A and rotenone. PMID:6061

  18. Kinetics characterization of c-Src binding to lipid membranes: Switching from labile to persistent binding.

    PubMed

    Le Roux, Anabel-Lise; Busquets, Maria Antònia; Sagués, Francesc; Pons, Miquel

    2016-02-01

    Cell signaling by the c-Src proto-oncogen requires the attachment of the protein to the inner side of the plasma membrane through the myristoylated N-terminal region, known as the SH4 domain. Additional binding regions of lower affinity are located in the neighbor intrinsically disordered Unique domain and the structured SH3 domain. Here we present a surface plasmon resonance study of the binding of a myristoylated protein including the SH4, Unique and SH3 domains of c-Src to immobilized liposomes. Two distinct binding processes were observed: a fast and a slow one. The second process lead to a persistently bound form (PB) with a slower binding and a much slower dissociation rate than the first one. The association and dissociation of the PB form could be detected using an anti-SH4 antibody. The kinetic analysis revealed that binding of the PB form follows a second order rate law suggesting that it involves the formation of c-Src dimers on the membrane surface. A kinetically equivalent PB form is observed in a myristoylated peptide containing only the SH4 domain but not in a construct including the three domains but with a 12-carbon lauroyl substituent instead of the 14-carbon myristoyl group. The PB form is observed with neutral lipids but its population increases when the immobilized liposomes contain negatively charged lipids. We suggest that the PB form may represent the active signaling form of c-Src while the labile form provides the capacity for fast 2D search of the target signaling site on the membrane surface. PMID:26638178

  19. High throughput adjustable 96-well plate assay for androgen receptor binding: a practical approach for EDC screening using the chimpanzee AR.

    PubMed

    Hartig, P C; Cardon, M C; Blystone, C R; Gray, L E; Wilson, V S

    2008-09-26

    The issue as to whether natural and man-made chemicals interfere with endocrine function has raised concerns. This interference could be biologically significant even at very low doses if the chemicals interact deleteriously with hormone receptors at low concentrations. Therefore, the United States Environmental Protection Agency (USEPA) Office of Coordination and Policy (OSCP) requested that a nonhuman mammalian androgen receptor binding assay be developed for possible use in their Endocrine Disruptor Screening Program (EDSP). Ideally, this assay would be high throughput, not use animals as a source of receptor protein, easily deployed throughout the scientific community, utilize reagents available to both the public and private sector, and have the potential for future automation. We developed a highly modified 96-well plate assay which meets these criteria. It employs a baculovirus expressed recombinant primate androgen receptor which is publically available and exploits the unique ability of some mammalian androgen receptors to remain biologically active after guanidine hydrochloride (GdnHCl) solubilization. This GdnHCl treated receptor remains soluble and requires no additional purification prior to use. We provide a very detailed description of the assay protocol itself, and similarly detailed method for producing and solubilizing the receptor. PMID:18691642

  20. NPA binding activity is peripheral to the plasma membrane and is associated with the cytoskeleton.

    PubMed Central

    Cox, D N; Muday, G K

    1994-01-01

    N-1-Naphthylphthalamic acid (NPA) binding activity is released into the supernatant when plasma membranes are subjected to high-salt treatment, indicating that this activity is peripherally associated with the membrane. Extraction of plasma membrane vesicles with Triton X-100 resulted in retention of NPA binding activity in the detergent-insoluble cytoskeletal pellet. Treatment of this pellet with KI released NPA binding activity, actin, and alpha-tubulin. Dialysis to remove KI led to the repolymerization of cytoskeletal elements and movement of NPA binding activity into an insoluble cytoskeletal pellet. NPA binding activity partitioned into the detergent-insoluble cytoskeletal pellet obtained from both zucchini and maize membranes and was released from these pellets by KI treatment. Treatment of a cytoskeletal pellet with cytochalasin B doubled NPA binding activity in the resulting supernatant. Together, these experiments indicate that NPA binding activity is peripherally associated with the plasma membrane and interacts with the cytoskeleton in vitro. PMID:11536654

  1. Reconstitution into liposomes of membrane proteins involved in ribosome binding on rough endoplasmic reticulum. Ribosome-binding capacity.

    PubMed Central

    Yamaguchi, M; Sakai, M; Horigome, T; Omata, S; Sugano, H

    1981-01-01

    A membrane protein fraction having a high affinity for polyribosomes was isolated from microsomal membranes of rat liver and was incorporated into liposomes made from microsomal lipids to evaluate the polyribosome-binding capacity of the reconstituted liposomes, with the following results. (1) The polyribosome binding to the reconstituted liposomes depended on the amounts of polyribosomes added to the binding mixture. (2) Liposomes made from lipids alone did not bind any polyribosomes. (3) The polyribosome-binding capacity of the reconstituted liposomes was very sensitive to proteolytic enzyme and strongly inhibited by addition of 0.1 mM-aurintricarboxylic acid or by increasing KCl concentration. These results suggest that the binding mechanism of polyribosomes to the reconstituted liposomes is much like that for rough microsomal membrane stripped of endogenous polyribosomes. PMID:7306032

  2. Uncoupling the Structure-Activity Relationships of β2 Adrenergic Receptor Ligands from Membrane Binding.

    PubMed

    Dickson, Callum J; Hornak, Viktor; Velez-Vega, Camilo; McKay, Daniel J J; Reilly, John; Sandham, David A; Shaw, Duncan; Fairhurst, Robin A; Charlton, Steven J; Sykes, David A; Pearlstein, Robert A; Duca, Jose S

    2016-06-23

    Ligand binding to membrane proteins may be significantly influenced by the interaction of ligands with the membrane. In particular, the microscopic ligand concentration within the membrane surface solvation layer may exceed that in bulk solvent, resulting in overestimation of the intrinsic protein-ligand binding contribution to the apparent/measured affinity. Using published binding data for a set of small molecules with the β2 adrenergic receptor, we demonstrate that deconvolution of membrane and protein binding contributions allows for improved structure-activity relationship analysis and structure-based drug design. Molecular dynamics simulations of ligand bound membrane protein complexes were used to validate binding poses, allowing analysis of key interactions and binding site solvation to develop structure-activity relationships of β2 ligand binding. The resulting relationships are consistent with intrinsic binding affinity (corrected for membrane interaction). The successful structure-based design of ligands targeting membrane proteins may require an assessment of membrane affinity to uncouple protein binding from membrane interactions. PMID:27239696

  3. Synthesis of novel C17 steroidal carbamates. Studies on CYP17 action, androgen receptor binding and function, and prostate cancer cell growth.

    PubMed

    Moreira, Vânia M A; Vasaitis, Tadas S; Guo, Zhiyong; Njar, Vincent C O; Salvador, Jorge A R

    2008-11-01

    We have exploited the reaction of 1,1'-carbonylbis(2-methylimidazole) (CBMI) with several 17beta-hydroxy androstanes to synthesize a series of novel C17 steroidal carbamates. Structural elucidation features have been provided for the final compounds based on 1D and 2D NMR techniques, IR spectroscopy, and related literature. The new compounds were tested for inhibition of human cytochrome 17alpha-hydroxylase-C17,20-lyase (CYP17) and androgen receptor (AR) binding and function effects. Their inhibitory potential against PC-3 cell proliferation was also evaluated. Compounds 11 and 23 were found to inhibit CYP17 with IC50 values of 17.1 and 11.5 microM, respectively. The carbamate moiety at C17 allowed tight binding of the synthesized compounds to both wild-type (wt-) and mutated AR. When bound to the mutated AR, the compounds were found to have a dual effect, stimulating transcription at low concentrations while almost fully blocking it at the higher concentrations tested, in the presence of the natural androgen dihydrotestosterone (DHT). Compounds 8 and 12 were the most active against PC-3 cell proliferation with EC50 values of 2.2 and 0.2 microM, respectively. PMID:18582482

  4. Optimization of polyvinylidene fluoride (PVDF) membrane fabrication for protein binding using statistical experimental design.

    PubMed

    Ahmad, A L; Ideris, N; Ooi, B S; Low, S C; Ismail, A

    2016-01-01

    Statistical experimental design was employed to optimize the preparation conditions of polyvinylidenefluoride (PVDF) membranes. Three variables considered were polymer concentration, dissolving temperature, and casting thickness, whereby the response variable was membrane-protein binding. The optimum preparation for the PVDF membrane was a polymer concentration of 16.55 wt%, a dissolving temperature of 27.5°C, and a casting thickness of 450 µm. The statistical model exhibits a deviation between the predicted and actual responses of less than 5%. Further characterization of the formed PVDF membrane showed that the morphology of the membrane was in line with the membrane-protein binding performance. PMID:27088961

  5. Binding kinetics of membrane-anchored receptors and ligands: Molecular dynamics simulations and theory

    NASA Astrophysics Data System (ADS)

    Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard; Weikl, Thomas R.

    2015-12-01

    The adhesion of biological membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. Central questions are how the binding kinetics of these proteins is affected by the membranes and by the membrane anchoring of the proteins. In this article, we (i) present detailed data for the binding of membrane-anchored proteins from coarse-grained molecular dynamics simulations and (ii) provide a theory that describes how the binding kinetics depends on the average separation and thermal roughness of the adhering membranes and on the anchoring, lengths, and length variations of the proteins. An important element of our theory is the tilt of bound receptor-ligand complexes and transition-state complexes relative to the membrane normals. This tilt results from an interplay of the anchoring energy and rotational entropy of the complexes and facilitates the formation of receptor-ligand bonds at membrane separations smaller than the preferred separation for binding. In our simulations, we have considered both lipid-anchored and transmembrane receptor and ligand proteins. We find that the binding equilibrium constant and binding on-rate constant of lipid-anchored proteins are considerably smaller than the binding constant and on-rate constant of rigid transmembrane proteins with identical binding domains.

  6. Repression of Runx2 by Androgen Receptor (AR) in Osteoblasts and Prostate Cancer Cells: AR Binds Runx2 and Abrogates Its Recruitment to DNA

    PubMed Central

    Baniwal, Sanjeev K.; Khalid, Omar; Sir, Donna; Buchanan, Grant; Coetzee, Gerhard A.; Frenkel, Baruch

    2009-01-01

    Runx2 and androgen receptor (AR) are master transcription factors with pivotal roles in bone metabolism and prostate cancer (PCa). We dissected AR-mediated repression of Runx2 in dihydrotestosterone (DHT)-treated osteoblastic and PCa cells using reporter assays and endogenous Runx2 target genes. Repression required DHT, but not AR’s transactivation function, and was associated with nuclear colocalization of the two proteins. Runx2 and AR coimmunoprecipitated and interacted directly in glutathione-S-transferase pull-down assays. Interaction was ionic in nature. Intact AR DNA-binding domain (DBD) was necessary and sufficient for both interaction with Runx2 and its repression. Runx2 sequences required for interaction were the C-terminal 132 amino acid residues together with the Runt DBD. Runx2 DNA binding was abrogated by endogenous AR in chromatin immunoprecipitation assays and by recombinant AR-DBD in gel shift assays. Furthermore, AR caused increased nuclear mobility of Runx2 as indicated by faster fluorescence recovery after photobleaching. Thus, AR binds Runx2 and abrogates its binding to DNA and possibly to other nuclear components. Clinical relevance of our results was suggested by an inverse correlation between expression of AR-responsive prostate-specific antigen and osteocalcin genes in PCa biopsies. Given the tumor suppressor properties of Runx2, its repression by AR may constitute a mechanism of hormone carcinogenesis. Attenuation of Runx2 by AR in osteoblasts may play a role in skeletal metabolism: the bone-sparing effect of androgens is attributable, in part, to keeping Runx2 activity in check and preventing high-turnover bone disease such as seen after castration and in transgenic mice overexpressing Runx2 in osteoblasts. PMID:19389811

  7. Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase

    PubMed Central

    De Michelis, Maria Ida; Pugliarello, Maria Chiara; Rasi-Caldogno, Franca

    1989-01-01

    The characteristics of fusicoccin binding were investigated in microsomes from 24-h-old radish (Raphanus sativus L.) seedlings. The time course of fusicoccin binding depended on fusicoccin concentration: equilibrium was reached much faster at 10 nanomolar fusicoccin than at 0.3 nanomolar fusicoccin. Scatchard analysis of equilibrium binding as a function of fusicoccin concentration indicated a single class of receptor sites with a Kd of 1.8 nanomolar and a site density of 6.3 picomoles per milligram protein. Similar values (Kd 1.7 nanomolar and site density 7 picomoles per milligram protein) were obtained from the analysis of the dependence of equilibrium binding on membrane concentration at fixed fusicoccin concentrations. Fusicoccin binding comigrated with the plasma membrane H+-ATPase in an equilibrium sucrose density gradient: both activities formed a sharp peak (1.18 grams per milliliter) clearly distinct from that of markers of other membranes which all peaked at lower densities. The saturation profiles of fusicoccin binding and of fusicoccin-induced activation of the plasma membrane H+-ATPase, measured under identical conditions, were similar, supporting the view that fusicoccin-induced activation of the plasma membrane H+-ATPase is mediated by fusicoccin binding to its plasma membrane receptor. PMID:16666723

  8. Evaluation of OASIS QSAR Models Using ToxCast™ in Vitro Estrogen and Androgen Receptor Binding Data and Application in an Integrated Endocrine Screening Approach

    PubMed Central

    Bhhatarai, Barun; Wilson, Daniel M.; Price, Paul S.; Marty, Sue; Parks, Amanda K.; Carney, Edward

    2016-01-01

    Background: Integrative testing strategies (ITSs) for potential endocrine activity can use tiered in silico and in vitro models. Each component of an ITS should be thoroughly assessed. Objectives: We used the data from three in vitro ToxCast™ binding assays to assess OASIS, a quantitative structure-activity relationship (QSAR) platform covering both estrogen receptor (ER) and androgen receptor (AR) binding. For stronger binders (described here as AC50 < 1 μM), we also examined the relationship of QSAR predictions of ER or AR binding to the results from 18 ER and 10 AR transactivation assays, 72 ER-binding reference compounds, and the in vivo uterotrophic assay. Methods: NovaScreen binding assay data for ER (human, bovine, and mouse) and AR (human, chimpanzee, and rat) were used to assess the sensitivity, specificity, concordance, and applicability domain of two OASIS QSAR models. The binding strength relative to the QSAR-predicted binding strength was examined for the ER data. The relationship of QSAR predictions of binding to transactivation- and pathway-based assays, as well as to in vivo uterotrophic responses, was examined. Results: The QSAR models had both high sensitivity (> 75%) and specificity (> 86%) for ER as well as both high sensitivity (92–100%) and specificity (70–81%) for AR. For compounds within the domains of the ER and AR QSAR models that bound with AC50 < 1 μM, the QSAR models accurately predicted the binding for the parent compounds. The parent compounds were active in all transactivation assays where metabolism was incorporated and, except for those compounds known to require metabolism to manifest activity, all assay platforms where metabolism was not incorporated. Compounds in-domain and predicted to bind by the ER QSAR model that were positive in ToxCast™ ER binding at AC50 < 1 μM were active in the uterotrophic assay. Conclusions: We used the extensive ToxCast™ HTS binding data set to show that OASIS ER and AR QSAR models had

  9. Control of adrenal androgen production.

    PubMed

    Odell, W D; Parker, L N

    The major adrenal androgens are dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEAS) and androstenedione (delta 4). Studies by Cutler et al in 1978 demonstrated that these androgens are detectable in blood of all domestic and laboratory animals studied, but that only 4 species show increase in one or more with sexual maturation: rabbit, dog, chimpanzee and man. Studies by Grover and Odell in 1975 show these androgens do not bind to the androgen receptor obtained from rat prostate and thus probably are androgens only by conversion to an active androgen in vivo. Thomas and Oake in 1974 showed human skin converted DHEA to testosterone. The control of adrenal androgen secretion is in part modulated by ACTH. However, other factors or hormones must exist also, for a variety of clinical observations show dissociation in adrenal androgen versus cortisol secretion. Other substances that have been said to be controllers of adrenal androgen secretion include estrogens, prolactin, growth hormone, gonadotropins and lipotropin. None of these appear to be the usual physiological modulator, although under some circumstances each may increase androgen production. Studies from our laboratory using in vivo experiments in the castrate dog and published in 1979 indicated that crude extracts of bovine pituitary contained a substance that either modified ACTH stimulation of adrenal androgen secretion, or stimulated secretion itself - Cortisol Androgen Stimulating Hormone. Parker et al in 1983 showed a 60,000 MW glycoprotein was extractable from human pituitaries, which stimulated DHA secretion by dispersed canine adrenal cells in vitro, but did not stimulate cortisol secretion. This material contained no ACTH by radioimmunoassay. In 1982 Brubaker et al reported a substance was also present in human fetal pituitaries, which stimulated DHA secretion, but did not effect cortisol. PMID:6100259

  10. Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells

    NASA Astrophysics Data System (ADS)

    Wang, Wei; Yang, Yunze; Wang, Shaopeng; Nagaraj, Vinay J.; Liu, Qiang; Wu, Jie; Tao, Nongjian

    2012-10-01

    Membrane proteins mediate a variety of cellular responses to extracellular signals. Although membrane proteins are studied intensively for their values as disease biomarkers and therapeutic targets, in situ investigation of the binding kinetics of membrane proteins with their ligands has been a challenge. Traditional approaches isolate membrane proteins and then study them ex situ, which does not reflect accurately their native structures and functions. We present a label-free plasmonic microscopy method to map the local binding kinetics of membrane proteins in their native environment. This analytical method can perform simultaneous plasmonic and fluorescence imaging, and thus make it possible to combine the strengths of both label-based and label-free techniques in one system. Using this method, we determined the distribution of membrane proteins on the surface of single cells and the local binding kinetic constants of different membrane proteins. Furthermore, we studied the polarization of the membrane proteins on the cell surface during chemotaxis.

  11. Effect of membrane protein concentration on binding of /sup 3/H-imipramine in human platelets

    SciTech Connect

    Barkai, A.I.; Kowalik, S.; Baron, M.

    1985-02-01

    Binding of /sup 3/H-imipramine to platelet membranes has been implicated as a marker for depression. Comparing /sup 3/H-IMI binding between depressed patients and normal subjects we observed an increase in the dissociation constant Kd with increasing membrane protein. This phenomenon was studied more rigorously in five normal subjects. Platelet membranes were prepared and adjusted to four concentrations of protein ranging from 100 to 800 micrograms/ml. The /sup 3/H-IMI binding parameters of maximum binding sites number (Bmax) and Kd were obtained by Scatchard analysis at each membrane concentration. A positive linear relationship was found between K/sub d/ values and the concentration of membrane protein in the assay, but no change was observed in Bmax. The variability in Kd values reported in the literature may be accounted for in part by the different concentrations of membrane protein used in various studies.

  12. Engineering a Highly Hydrophilic PVDF Membrane via Binding TiO₂Nanoparticles and a PVA Layer onto a Membrane Surface.

    PubMed

    Qin, Aiwen; Li, Xiang; Zhao, Xinzhen; Liu, Dapeng; He, Chunju

    2015-04-29

    A highly hydrophilic PVDF membrane was fabricated through chemically binding TiO2 nanoparticles and a poly(vinyl alcohol) (PVA) layer onto a membrane surface simultaneously. The chemical composition of the modified membrane surface was determined by X-ray photoelectron spectroscopy, and the binding performance of TiO2 nanoparticles and the PVA layer was investigated by a rinsing test. The results indicated that the TiO2 nanoparticles were uniformly and strongly tailored onto the membrane surface, while the PVA layer was firmly attached onto the surface of TiO2 nanoparticles and the membrane by adsorption-cross-linking. The possible mechanisms during the modification process and filtration performance, i.e., water permeability and bovine serum albumin (BSA) rejection, were investigated as well. Furthermore, antifouling property was discussed through multicycles of BSA solution filtration tests, where the flux recovery ratio was significantly increased from 20.0% for pristine PVDF membrane to 80.5% for PVDF/TiO2/PVA-modified membrane. This remarkable promotion is mainly ascribed to the improvement of surface hydrophilicity, where the water contact angle of the membrane surface was decreased from 84° for pristine membrane to 24° for PVDF/TiO2/PVA membrane. This study presents a novel and varied strategy for immobilization of nanoparticles and PVA layer on substrate surface, which could be easily adapted for a variety of materials for surface modification. PMID:25806418

  13. Monolayers of a Model Anesthetic-Binding Membrane Protein: Formation, Characterization, and Halothane-Binding Affinity

    PubMed Central

    Churbanova, Inna Y.; Tronin, Andrey; Strzalka, Joseph; Gog, Thomas; Kuzmenko, Ivan; Johansson, Jonas S.; Blasie, J. Kent

    2006-01-01

    hbAP0 is a model membrane protein designed to possess an anesthetic-binding cavity in its hydrophilic domain and a cation channel in its hydrophobic domain. Grazing incidence x-ray diffraction shows that hbAP0 forms four-helix bundles that are vectorially oriented within Langmuir monolayers at the air-water interface. Single monolayers of hbAP0 on alkylated solid substrates would provide an optimal system for detailed structural and dynamical studies of anesthetic-peptide interaction via x-ray and neutron scattering and polarized spectroscopic techniques. Langmuir-Blodgett and Langmuir-Schaeffer deposition and self-assembly techniques were used to form single monolayer films of the vectorially oriented peptide hbAP0 via both chemisorption and physisorption onto suitably alkylated solid substrates. The films were characterized by ultraviolet absorption, ellipsometry, circular dichroism, and polarized Fourier transform infrared spectroscopy. The α-helical secondary structure of the peptide was retained in the films. Under certain conditions, the average orientation of the helical axis was inclined relative to the plane of the substrate, approaching perpendicular in some cases. The halothane-binding affinity of the vectorially oriented hbAP0 peptide in the single monolayers, with the volatile anesthetic introduced into the moist vapor environment of the monolayer, was found to be similar to that for the detergent-solubilized peptide. PMID:16473900

  14. MBPpred: Proteome-wide detection of membrane lipid-binding proteins using profile Hidden Markov Models.

    PubMed

    Nastou, Katerina C; Tsaousis, Georgios N; Papandreou, Nikos C; Hamodrakas, Stavros J

    2016-07-01

    A large number of modular domains that exhibit specific lipid binding properties are present in many membrane proteins involved in trafficking and signal transduction. These domains are present in either eukaryotic peripheral membrane or transmembrane proteins and are responsible for the non-covalent interactions of these proteins with membrane lipids. Here we report a profile Hidden Markov Model based method capable of detecting Membrane Binding Proteins (MBPs) from information encoded in their amino acid sequence, called MBPpred. The method identifies MBPs that contain one or more of the Membrane Binding Domains (MBDs) that have been described to date, and further classifies these proteins based on their position in respect to the membrane, either as peripheral or transmembrane. MBPpred is available online at http://bioinformatics.biol.uoa.gr/MBPpred. This method was applied in selected eukaryotic proteomes, in order to examine the characteristics they exhibit in various eukaryotic kingdoms and phyla. PMID:27048983

  15. Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry.

    PubMed

    Cong, Xiao; Liu, Yang; Liu, Wen; Liang, Xiaowen; Russell, David H; Laganowsky, Arthur

    2016-04-01

    Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites. PMID:27015007

  16. NMR WaterLOGSY Reveals Weak Binding of Bisphenol A with Amyloid Fibers of a Conserved 11 Residue Peptide from Androgen Receptor.

    PubMed

    Asencio-Hernández, Julia; Kieffer, Bruno; Delsuc, Marc-André

    2016-01-01

    There is growing evidence that bisphenol A (BPA), a molecule largely released in the environment, has detrimental effects on ecosystems and on human health. It acts as an endocrine disruptor targeting steroid hormone receptors, such as the estrogen receptor (ER), estrogen-related receptor (ERR) and androgen receptor (AR). BPA-derived molecules have recently been shown to interact with the AR N-terminal domain (AR-NTD), which is known to be largely intrinsically disordered. This N-terminal domain contains an 11 residue conserved domain that forms amyloid fibers upon oxidative dimerisation through its strictly conserved Cys240 residue. We investigate here the interaction of BPA, and other potential endocrine disruptors, with AR-NTD amyloid fibers using the WaterLOGSY NMR experiment. We observed a selective binding of these compounds to the amyloid fibers formed by the AR-NTD conserved region and glutamine homopolymers. This observation suggests that the high potency of endocrine disruptors may result, in part, from their ability to bind amyloid forms of nuclear receptors in addition to their cognate binding sites. This property may be exploited to design future therapeutic strategies targeting AR related diseases such as the spinal bulbar muscular atrophy or prostate cancer. The ability of NMR WaterLOGSY experiments to detect weak interactions between small ligands and amyloid fibers may prove to be of particular interest for identifying promising hit molecules. PMID:27583469

  17. Membrane Binding and Modulation of the PDZ Domain of PICK1

    PubMed Central

    Erlendsson, Simon; Madsen, Kenneth Lindegaard

    2015-01-01

    Scaffolding proteins serve to assemble protein complexes in dynamic processes by means of specific protein-protein and protein-lipid binding domains. Many of these domains bind either proteins or lipids exclusively; however, it has become increasingly evident that certain domains are capable of binding both. Especially, many PDZ domains, which are highly abundant protein-protein binding domains, bind lipids and membranes. Here we provide an overview of recent large-scale studies trying to generalize and rationalize the binding patterns as well as specificity of PDZ domains towards membrane lipids. Moreover, we review how these PDZ-membrane interactions are regulated in the case of the synaptic scaffolding protein PICK1 and how this might affect cellular localization and function. PMID:26501328

  18. Deconstructing the DGAT1 Enzyme: Membrane Interactions at Substrate Binding Sites

    PubMed Central

    Lopes, Jose L. S.; Beltramini, Leila M.; Wallace, Bonnie A.; Araujo, Ana P. U.

    2015-01-01

    Diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates. PMID:25719207

  19. Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes.

    PubMed

    Olety, Balaji; Veatch, Sarah L; Ono, Akira

    2016-01-01

    The structural protein of HIV-1, Pr55(Gag) (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an essential step for virus particle formation, since a defect in Gag membrane binding results in severe impairment of viral particle production. To gain mechanistic details of Gag-lipid membrane interactions, in vitro methods based on NMR, protein footprinting, surface plasmon resonance, liposome flotation centrifugation, or fluorescence lipid bead binding have been developed thus far. However, each of these in vitro methods has its limitations. To overcome some of these limitations and provide a complementary approach to the previously established methods, we developed an in vitro assay in which interactions between HIV-1 Gag and lipid membranes take place in a "cell-like" environment. In this assay, Gag binding to lipid membranes is visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique. Here we describe the background and the protocols to obtain myristoylated full-length Gag proteins and GUV membranes necessary for the assay and to detect Gag-GUV binding by microscopy. PMID:27500610

  20. Receptor Concentration and Diffusivity Control Multivalent Binding of Sv40 to Membrane Bilayers

    PubMed Central

    Szklarczyk, Oliwia M.; González-Segredo, Nélido; Kukura, Philipp; Oppenheim, Ariella; Choquet, Daniel; Sandoghdar, Vahid; Helenius, Ari; Sbalzarini, Ivo F.; Ewers, Helge

    2013-01-01

    Abstract Incoming Simian Virus 40 particles bind to their cellular receptor, the glycolipid GM1, in the plasma membrane and thereby induce membrane deformation beneath the virion leading to endocytosis and infection. Efficient membrane deformation depends on receptor lipid structure and the organization of binding sites on the internalizing particle. To determine the role of receptor diffusion, concentration and the number of receptors required for stable binding in this interaction, we analyze the binding of SV40 to GM1 in supported membrane bilayers by computational modeling based on experimental data. We measure the diffusion rates of SV40 virions in solution by fluorescence correlation spectroscopy and of the receptor in bilayers by single molecule tracking. Quartz-crystal microbalance with dissipation (QCM-D) is used to measure binding of SV40 virus-like particles to bilayers containing the viral receptor GM1. We develop a phenomenological stochastic dynamics model calibrated against this data, and use it to investigate the early events of virus attachment to lipid membranes. Our results indicate that SV40 requires at least 4 attached receptors to achieve stable binding. We moreover find that receptor diffusion is essential for the establishment of stable binding over the physiological range of receptor concentrations and that receptor concentration controls the mode of viral motion on the target membrane. Our results provide quantitative insight into the initial events of virus-host interaction at the nanoscopic level. PMID:24244125

  1. Dissociation of membrane binding and lytic activities of the lymphocyte pore-forming protein (perforin).

    PubMed

    Young, J D; Damiano, A; DiNome, M A; Leong, L G; Cohn, Z A

    1987-05-01

    Granules isolated from CTL and NK cells contain a cytolytic pore-forming protein (PFP/perforin). At low temperatures (on ice), PFP binds to erythrocyte membranes without producing hemolysis. Hemolysis occurs when the PFP-bound erythrocytes are warmed up to 37 degrees C, which defines a temperature-dependent, lytic (pore-formation) step distinct from the membrane-binding event. Ca2+ and neutral pH are required for both membrane binding and pore formation by PFP. Serum, LDL, HDL, and heparin inhibit the hemolytic activity of PFP by blocking its binding to lipid membranes. Lysis by PFP that has bound to erythrocyte membranes is no longer susceptible to the effect of these inhibitors. The hemolytic activities associated with intact granules and solubilized PFP show different requirements for Ca2+ and pH, indicating that cytolysis produced by isolated granules may involve an additional step, possibly fusion of granules with membranes. It is suggested that three distinct Ca2+- and pH-dependent events may be involved during cell killing by CTL and NK cells: fusion of cytoplasmic granules of effector cells with their plasma membrane, releasing PFP from cells; binding of the released PFP to target membranes; and insertion of monomers and the subsequent formation of lytic pores in the target membrane. The serum-mediated inhibition of membrane binding by PFP could prevent the accidental injury of bystander cells by cell-released PFP, but would allow cytolysis to proceed to completion once PFP has bound to the target membrane. PMID:3494808

  2. Ambidextrous Binding of Cell and Membrane Bilayers by Soluble Matrix Metalloproteinase-12

    PubMed Central

    Koppisetti, Rama K.; Fulcher, Yan G.; Jurkevich, Alexander; Prior, Stephen H.; Xu, Jia; Lenoir, Marc; Overduin, Michael; Van Doren, Steven R.

    2014-01-01

    Matrix metalloproteinases (MMPs) regulate tissue remodeling, inflammation, and disease progression. Some soluble MMPs are inexplicably active near cell surfaces. Here, we demonstrate binding of MMP-12 directly to bilayers and cellular membranes using paramagnetic NMR and fluorescence. Opposing sides of the catalytic domain engage spin-labeled membrane mimics. Loops project from the β-sheet interface to contact the phospholipid bilayer with basic and hydrophobic residues. The distal membrane interface comprises loops on the other side of the catalytic cleft. Both interfaces mediate MMP-12 association with vesicles and cell membranes. MMP-12 binds plasma membranes and is internalized to hydrophobic perinuclear features, the nuclear membrane, and inside the nucleus within minutes. While binding of TIMP-2 to MMP-12 hinders membrane interactions beside the active site, TIMP-2-inhibited MMP-12 binds vesicles and cells, suggesting compensatory rotation of its membrane approaches. MMP-12 association with diverse cell membranes may target its activities to modulate innate immune responses and inflammation. PMID:25412686

  3. Ambidextrous binding of cell and membrane bilayers by soluble matrix metalloproteinase-12.

    PubMed

    Koppisetti, Rama K; Fulcher, Yan G; Jurkevich, Alexander; Prior, Stephen H; Xu, Jia; Lenoir, Marc; Overduin, Michael; Van Doren, Steven R

    2014-01-01

    Matrix metalloproteinases (MMPs) regulate tissue remodelling, inflammation and disease progression. Some soluble MMPs are inexplicably active near cell surfaces. Here we demonstrate the binding of MMP-12 directly to bilayers and cellular membranes using paramagnetic NMR and fluorescence. Opposing sides of the catalytic domain engage spin-labelled membrane mimics. Loops project from the β-sheet interface to contact the phospholipid bilayer with basic and hydrophobic residues. The distal membrane interface comprises loops on the other side of the catalytic cleft. Both interfaces mediate MMP-12 association with vesicles and cell membranes. MMP-12 binds plasma membranes and is internalized to hydrophobic perinuclear features, the nuclear membrane and inside the nucleus within minutes. While binding of TIMP-2 to MMP-12 hinders membrane interactions beside the active site, TIMP-2-inhibited MMP-12 binds vesicles and cells, suggesting compensatory rotation of its membrane approaches. MMP-12 association with diverse cell membranes may target its activities to modulate innate immune responses and inflammation. PMID:25412686

  4. Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes.

    PubMed Central

    Pestonjamasp, K; Amieva, M R; Strassel, C P; Nauseef, W M; Furthmayr, H; Luna, E J

    1995-01-01

    Actin-binding proteins in bovine neutrophil plasma membranes were identified using blot overlays with 125I-labeled F-actin. Along with surface-biotinylated proteins, membranes were enriched in major actin-binding polypeptides of 78, 81, and 205 kDa. Binding was specific for F-actin because G-actin did not bind. Further, unlabeled F-actin blocked the binding of 125I-labeled F-actin whereas other acidic biopolymers were relatively ineffective. Binding also was specifically inhibited by myosin subfragment 1, but not by CapZ or plasma gelsolin, suggesting that the membrane proteins, like myosin, bind along the sides of the actin filaments. The 78- and 81-kDa polypeptides were identified as moesin and ezrin, respectively, by co-migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with antibodies specific for moesin and ezrin. Although not present in detectable amounts in bovine neutrophils, radixin (a third and closely related member of this gene family) also bound 125I-labeled F-actin on blot overlays. Experiments with full-length and truncated bacterial fusion proteins localized the actin-binding site in moesin to the extreme carboxy terminus, a highly conserved sequence. Immunofluorescence micrographs of permeabilized cells and cell "footprints" showed moesin co-localization with actin at the cytoplasmic surface of the plasma membrane, consistent with a role as a membrane-actin-linking protein. Images PMID:7612961

  5. Dual modes of membrane binding direct pore formation by Streptolysin O.

    PubMed

    Mozola, Cara C; Caparon, Michael G

    2015-09-01

    Effector translocation is central to the virulence of many bacterial pathogens, including Streptococcus pyogenes, which utilizes the cholesterol-dependent cytolysin Streptolysin O (SLO) to translocate the NAD(+) glycohydrolase SPN into host cells during infection. SLO's translocation activity does not require host cell membrane cholesterol or pore formation by SLO, yet SLO does form pores during infection via a cholesterol-dependent mechanism. Although cholesterol was considered the primary receptor for SLO, SLO's membrane-binding domain also encodes a putative carbohydrate-binding site, implicating a potential glycan receptor in binding and pore formation. Analysis of carbohydrate-binding site SLO mutants and carbohydrate-defective cell lines revealed that glycan recognition is involved in SLO's pore formation pathway and is an essential step when SLO is secreted by non-adherent bacteria, as occurs during lysis of erythrocytes. However, SLO also recognizes host cell membranes via a second mechanism when secreted from adherent bacteria, which requires co-secretion of SPN but not glycan binding by SLO. This SPN-mediated membrane binding of SLO correlates with SPN translocation, and requires SPN's non-enzymatic domain, which is predicted to adopt the structure of a carbohydrate-binding module. SPN-dependent membrane binding also promotes pore formation by SLO, demonstrating that pore formation can occur by distinct pathways during infection. PMID:26059530

  6. Cooperative binding of Annexin A5 to phosphatidylserine on apoptotic cell membranes

    NASA Astrophysics Data System (ADS)

    Janko, Christina; Jeremic, Ivica; Biermann, Mona; Chaurio, Ricardo; Schorn, Christine; Muñoz, Luis E.; Herrmann, Martin

    2013-12-01

    Healthy cells exhibit an asymmetric plasma membrane with phosphatidylserine (PS) located on the cytoplasmic leaflet of the plasma membrane bilayer. Annexin A5-FITC, a PS binding protein, is commonly used to evaluate apoptosis in flow cytometry. PS exposed by apoptotic cells serves as a major ‘eat-me’ signal for phagocytes. Although exposition of PS has been observed after alternative stimuli, no clearance of viable, PS exposing cells has been detected. Thus, besides PS exposure, membranes of viable and apoptotic cells might exhibit specific characteristics. Here, we show that Annexin A5 binds in a cooperative manner to different types of dead cells. Shrunken apoptotic cells thereby showed the highest Hill coefficient values. Contrarily, parafomaldehyde fixation of apoptotic cells completely abrogates the cooperativity effect seen with dead and dying cells. We tend to speculate that the cooperative binding of Annexin A5 to the membranes of apoptotic cells reflects higher fluidity of the exposed membranes facilitating PS clustering.

  7. Evidence for multiple mechanisms for membrane binding and integration via carboxyl-terminal insertion sequences.

    PubMed

    Kim, P K; Janiak-Spens, F; Trimble, W S; Leber, B; Andrews, D W

    1997-07-22

    Subcellular localization of proteins with carboxyl-terminal insertion sequences requires the molecule be both targeted to and integrated into the correct membrane. The mechanism of membrane integration of cytochrome b5 has been shown to be promiscuous, spontaneous, nonsaturable, and independent of membrane proteins. Thus endoplasmic reticulum localization for cytochrome b5 depends primarily on accurate targeting to the appropriate membrane. Here direct comparison of this mechanism with that of three other proteins integrated into membranes via carboxyl-terminal insertion sequences [vesicle-associated membrane protein 1(Vamp1), polyomavirus middle-T antigen, and Bcl-2] revealed that, unlike cytochrome b5, membrane selectivity for these molecules is conferred at least in part by the mechanisms of membrane integration. Bcl-2 membrane integration was similar to that of cytochrome b5 except that insertion into lipid vesicles was inefficient. Unlike cytochrome b5 and Bcl-2, Vamp1 binding to canine pancreatic microsomes was saturable, ATP-dependent, and abolished by mild trypsin treatment of microsomes. Surprisingly, although the insertion sequence of polyomavirus middle-T antigen was sufficient to mediate electrostatic binding to membranes, binding did not lead to integration into the bilayer. Together these results demonstrate that there are at least two different mechanisms for correct membrane integration of proteins with insertion sequences, one mediated primarily by targeting and one relying on factors in the target membrane to mediate selective integration. Our results also demonstrate that, contrary to expectation, hydrophobicity is not sufficient for insertion sequence-mediated membrane integration. We suggest that the structure of the insertion sequence determines whether or not specific membrane-bound receptor proteins are required for membrane integration. PMID:9220974

  8. Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets

    PubMed Central

    Prada, Ilaria; Meldolesi, Jacopo

    2016-01-01

    Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated. PMID:27517914

  9. Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

    PubMed

    Prada, Ilaria; Meldolesi, Jacopo

    2016-01-01

    Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated. PMID:27517914

  10. Inactivation of Maize Leaf Phosphoenolpyruvate Carboxylase by the Binding to Chloroplast Membranes 1

    PubMed Central

    Wu, Min-Xian; Wedding, Randolph T.

    1992-01-01

    Phosphoenolpyruvate carboxylase (PEPC) purified from maize (Zea mays L.) leaves associates with maize leaf chloroplast membrane in vitro. The binding of PEPC to the membrane results in enzyme inactivation. A protein isolated from a maize leaf chloroplast membrane preparation inactivated PEPC. Treatment with membrane preparation or with partially purified inactivating protein accelerates PEPC inactivation at low temperature (4°C). Interaction of PEPC with chloroplast membrane or inactivating protein may inactivate the enzyme by influencing dissociation of the enzyme active tetramer. PMID:16652972

  11. The RNA-binding protein Sam68 regulates expression and transcription function of the androgen receptor splice variant AR-V7

    PubMed Central

    Stockley, Jacqueline; Markert, Elke; Zhou, Yan; Robson, Craig N.; Elliott, David J.; Lindberg, Johan; Leung, Hing Y.; Rajan, Prabhakar

    2015-01-01

    Castration-resistant (CR) prostate cancer (PCa) partly arises due to persistence of androgen receptor (AR) transcriptional activity in the absence of cognate ligand. An emerging mechanism underlying the CRPCa phenotype and predicting response to therapy is the expression of the constitutively-active AR-V7 splice variant generated by AR cryptic exon 3b inclusion. Here, we explore the role of the RNA-binding protein (RBP) Sam68 (encoded by KHDRBS1), which is over-expressed in clinical PCa, on AR-V7 expression and transcription function. Using a minigene reporter, we show that Sam68 controls expression of exon 3b resulting in an increase in endogenous AR-V7 mRNA and protein expression in RNA-binding-dependent manner. We identify a novel protein-protein interaction between Sam68 and AR-V7 mediated by a common domain shared with full-length AR, and observe these proteins in the cell nucleoplasm. Using a luciferase reporter, we demonstrate that Sam68 co-activates ligand-independent AR-V7 transcriptional activity in an RNA-binding-independent manner, and controls expression of the endogenous AR-V7-specific gene target UBE2C. Our data suggest that Sam68 has separable effects on the regulation of AR-V7 expression and transcriptional activity, through its RNA-binding capacity. Sam68 and other RBPs may control expression of AR-V7 and other splice variants as well as their downstream functions in CRPCa. PMID:26310125

  12. The RNA-binding protein Sam68 regulates expression and transcription function of the androgen receptor splice variant AR-V7.

    PubMed

    Stockley, Jacqueline; Markert, Elke; Zhou, Yan; Robson, Craig N; Elliott, David J; Lindberg, Johan; Leung, Hing Y; Rajan, Prabhakar

    2015-01-01

    Castration-resistant (CR) prostate cancer (PCa) partly arises due to persistence of androgen receptor (AR) transcriptional activity in the absence of cognate ligand. An emerging mechanism underlying the CRPCa phenotype and predicting response to therapy is the expression of the constitutively-active AR-V7 splice variant generated by AR cryptic exon 3b inclusion. Here, we explore the role of the RNA-binding protein (RBP) Sam68 (encoded by KHDRBS1), which is over-expressed in clinical PCa, on AR-V7 expression and transcription function. Using a minigene reporter, we show that Sam68 controls expression of exon 3b resulting in an increase in endogenous AR-V7 mRNA and protein expression in RNA-binding-dependent manner. We identify a novel protein-protein interaction between Sam68 and AR-V7 mediated by a common domain shared with full-length AR, and observe these proteins in the cell nucleoplasm. Using a luciferase reporter, we demonstrate that Sam68 co-activates ligand-independent AR-V7 transcriptional activity in an RNA-binding-independent manner, and controls expression of the endogenous AR-V7-specific gene target UBE2C. Our data suggest that Sam68 has separable effects on the regulation of AR-V7 expression and transcriptional activity, through its RNA-binding capacity. Sam68 and other RBPs may control expression of AR-V7 and other splice variants as well as their downstream functions in CRPCa. PMID:26310125

  13. Binding of rabbit muscle aldolase to band 3, the predominant polypeptide of the human erythrocyte membrane.

    PubMed

    Strapazon, E; Steck, T L

    1976-04-01

    Aldolase is a trace protein in isolated human red cell membrane preparations. Following total elution of the endogenous enzyme by a saline wash, the interaction of this membrane with rabbit muscle aldolase was studied. At saturation, exogenous aldolase constituted over 40% of the repleted membrane protein. Scatchard analysis revealed two classes of sites, each numbering approximately 7 X 10(5) per ghost. Specificity was suggested by the exclusive binding of the enzyme to the membrane's inner (cytoplasmic) surface. Furthermore, milimolar levels of fructose 1,6-bisphosphate eluted the enzyme from ghosts, while fructose 6-phosphate and NADH (a metabolite which elutes human erythrocyte glyceraldehyde-3-phosphate dehydrogenase (G3PD) from its binding site) were ineffectuve. Removing peripheral membrane proteins with EDTA and lithium 3,5-diiodosalicylate did not diminish the binding capacity of the membranes. An aldolase-band 3 complex, dissociable by high ionic strength or fructose 1,6-bisphosphate treatment, was demonstrated in Triton X-100 extracts of repleted membranes by rate zonal sedimentation analysis on sucrose gradients. We conclude that the association of rabbit muscle aldolase with isolated human erythrocyte membranes reflects its specific binding to band 3 at the cytoplasmic surface, as is also true of G3PD. PMID:1259946

  14. α-Synuclein and huntingtin exon 1 amyloid fibrils bind laterally to the cellular membrane

    PubMed Central

    Monsellier, Elodie; Bousset, Luc; Melki, Ronald

    2016-01-01

    Fibrillar aggregates involved in neurodegenerative diseases have the ability to spread from one cell to another in a prion-like manner. The underlying molecular mechanisms, in particular the binding mode of the fibrils to cell membranes, are poorly understood. In this work we decipher the modality by which aggregates bind to the cellular membrane, one of the obligatory steps of the propagation cycle. By characterizing the binding properties of aggregates made of α-synuclein or huntingtin exon 1 protein displaying similar composition and structure but different lengths to mammalian cells we demonstrate that in both cases aggregates bind laterally to the cellular membrane, with aggregates extremities displaying little or no role in membrane binding. Lateral binding to artificial liposomes was also observed by transmission electron microscopy. In addition we show that although α-synuclein and huntingtin exon 1 fibrils bind both laterally to the cellular membrane, their mechanisms of interaction differ. Our findings have important implications for the development of future therapeutic tools that aim to block protein aggregates propagation in the brain. PMID:26757959

  15. Identification of DNA-binding proteins on human umbilical vein endothelial cell plasma membrane.

    PubMed Central

    Chan, T M; Frampton, G; Cameron, J S

    1993-01-01

    The binding of anti-DNA antibodies to the endothelial cell is mediated through DNA, which forms a bridge between the immunoglobulin and the plasma membrane. We have shown that 32P-labelled DNA bound to the plasma membrane of human umbilical vein endothelial cells (HUVEC) by a saturable process, which could be competitively inhibited by non-radiolabelled DNA. In addition, DNA-binding was enhanced in HUVEC that had been treated with IL-1 alpha or tumour necrosis factor-alpha (TNF-alpha). DNA-binding proteins of mol. wt 46,000, 92,000, and 84,000 were identified by the binding of 32P-labelled DNA to plasma membrane proteins separated on SDS-PAGE. DNA-binding proteins of mol. wt 46,000 and 84,000 were also present in the cytosol and nucleus. Murine anti-DNA MoAb410 bound to a single band, at mol. wt 46,000, of plasma membrane protein, in the presence of DNA. Our results showed that DNA-binding proteins are present in different cellular fractions of endothelial cells. DNA-binding proteins on the cell membrane could participate in the in situ formation of immune deposits; and their presence in the cell nucleus suggests a potential role in the modulation of cell function. Images Fig. 3 Fig. 4 PMID:8419070

  16. Studies on membrane proteins involved in ribosome binding on the rough endoplasmic reticulum. Ribophorins have no ribosome-binding activity.

    PubMed Central

    Yoshida, H; Tondokoro, N; Asano, Y; Mizusawa, K; Yamagishi, R; Horigome, T; Sugano, H

    1987-01-01

    A membrane protein fraction showing affinity for ribosomes was isolated from rat liver microsomes (microsomal fractions) in association with ribosomes by treatment of the microsomes with Emulgen 913 and then solubilized from the ribosomes with sodium deoxycholate. This protein fraction was separated into two fractions, glycoproteins, including ribophorins I and II, and non-glycoproteins, virtually free from ribophorins I and II, on concanavalin A-Sepharose columns. The two fractions were each reconstituted into liposomes to determine their ribosome-binding activities. The specific binding activity of the non-glycoprotein fraction was approx. 2.3-fold higher than that of the glycoprotein fraction. The recovery of ribosome-binding capacity of the two fractions was about 85% of the total binding capacity of the material applied to a concanavalin A-Sepharose column, and about 90% of it was found in the non-glycoprotein fraction. The affinity constants of the ribosomes for the reconstituted liposomes were somewhat higher than those for stripped rough microsomes. The mode of ribosome binding to the reconstituted liposomes was very similar to that to the stripped rough microsomes, in its sensitivity to proteolytic enzymes and its strong inhibition by increasing KCl concentration. These results support the idea that ribosome binding to rat liver microsomes is not directly mediated by ribophorins I and II, but that another unidentified membrane protein(s) plays a role in ribosome binding. Images Fig. 1. Fig. 3. Fig. 5. PMID:3663192

  17. LIPID - BINDING SURFACES OF MEMBRANE PROTEINS: EVIDENCE FROM EVOLUTIONARY AND STRUCTURAL ANALYSIS

    PubMed Central

    Adamian, Larisa; Naveed, Hammad; Liang, Jie

    2011-01-01

    Membrane proteins function in the diverse environment of the lipid bilayer. Experimental evidence suggests that some lipid molecules bind tightly to specific sites on the membrane protein surface. These lipid molecules often act as co-factors and play important functional roles. In this study, we have assessed the evolutionary selection pressure experienced at lipid-binding sites in a set of α-helical and β-barrel membrane proteins using posterior probability analysis of the ratio of synonymous vs. nonsynonymous substitutions (ω-ratio). We have also carried out a geometric analysis of the membrane protein structures to identify residues in close contact with co-crystallized lipids. We found that residues forming cholesterol-binding sites in both β2-adrenergic receptor and Na+-K+-ATPase exhibit strong conservation, which can be characterized by an expanded cholesterol consensus motif for GPCRs. Our results suggest the functional importance of aromatic stacking interactions and interhelical hydrogen bonds in facilitating protein-cholesterol interactions, which is now reflected in the expanded motif. We also find that residues forming the cardiolipin-binding site in formate dehydrogenase-N γ-subunit and the phosphatidylglycerol binding site in KcsA are under strong purifying selection pressure. Although the lipopolysaccharide (LPS)-binding site in ferric hydroxamate uptake receptor (FhuA) is only weakly conserved, we show using a statistical mechanical model that LPS binds to the least stable FhuA β-strand and protects it from the bulk lipid. Our results suggest that specific lipid binding may be a general mechanism employed by β-barrel membrane proteins to stabilize weakly stable regions. Overall, we find that the residues forming specific lipid binding sites on the surfaces of membrane proteins often experience strong purifying selection pressure. PMID:21167813

  18. Lipoprotein receptors in copper-deficient rats: high density lipoprotein binding to liver membranes

    SciTech Connect

    Hassel, C.A.; Lei, K.Y.; Marchello, J.A.

    1986-03-05

    In copper-deficient rats, the observed hyperlipoproteinemia was mainly due to the elevation in high density lipoproteins (HDL). This study was designed to determine whether an impairment in the binding of HDL to liver membrane is responsible for the hyperlipoproteinemia. Sixty male Sprague-Dawley rats were randomly divided into 2 treatments, namely copper (Cu) deficient and adequate (less than 1 and 8 mg Cu/kg of diet). After 8 weeks, plasma, heart and liver tissues were obtained. Reduction in liver Cu content and elevation in heart to body weight ratio and plasma cholesterol confirmed that rats fed the test diet were Cu-deficient. Plasma HDL isolated from both Cu-deficient and control rats were iodinated and bound to liver membranes prepared from rats of each treatment. Binding of /sup 125/I-HDL was competitively inhibited by unlabelled rat HDL from both treatments, but not by human LDL. Scatchard analysis of specific binding data showed that maximal /sup 125/I-HDL binding (per mg membrane protein) to membranes prepared from Cu-deficient rats was not lower than controls. Furthermore, the amount of /sup 125/I-HDL from deficient rats specifically bound to liver membranes prepared from either treatment was not less than the amount of /sup 125/I-HDL from control rats bound to the same membranes. The data suggest that the hyperlipoproteinemia in Cu-deficient rats may not have resulted from a decrease in the number of hepatic HDL binding sites.

  19. Multi-scale molecular dynamics study of cholera pentamer binding to a GM1-phospholipid membrane.

    PubMed

    Sridhar, Akshay; Kumar, Amit; Dasmahapatra, Ashok Kumar

    2016-07-01

    The AB5 type toxin produced by the Vibrio cholerae bacterium is the causative agent of the cholera disease. The cholera toxin (CT) has been shown to bind specifically to GM1 glycolipids on the membrane surface. This binding of CT to the membrane is the initial step in its endocytosis and has been postulated to cause significant disruption to the membrane structure. In this work, we have carried out a combination of coarse-grain and atomistic simulations to study the binding of CT to a membrane modelled as an asymmetrical GM1-DPPC bilayer. Simulation results indicate that the toxin binds to the membrane through only three of its five B subunits, in effect resulting in a tilted bound configuration. Additionally, the binding of the CT can increase the area per lipid of GM1 leaflet, which in turn can cause the membrane regions interacting with the bound subunits to experience significant bilayer thinning and lipid tail disorder across both the leaflets. PMID:27474868

  20. The Role of Androgen and Androgen Receptor in the Skin-Related Disorders

    PubMed Central

    Lai, Jiann-Jyh; Chang, Philip; Lai, Kuo-Pao; Chen, Lumin; Chang, Chawnshang

    2013-01-01

    Androgen and androgen receptor (AR) may play important roles in several skin related diseases, such as androgenetic alopecia and acne vulgaris. Current treatments for these androgen/AR-involved diseases, which target the synthesis of androgens or prevent its binding to AR, can cause significant adverse side effects. Based on the recent studies using AR knockout mice, it has been suggested that AR and androgens play distinct roles in the skin pathogenesis, and AR seems to be a better target than androgens for the treatment of these skin diseases. Here we review recent studies of androgen/AR roles in several skin-related disorders, including acne vulgaris, androgenetic alopecia, and hirsutism, as well as cutaneous wound healing. PMID:22829074

  1. Lack of binding to isolated estrogen or androgen receptors, and inactivity in the immature rat uterotrophic assay, of the ultraviolet sunscreen filters Tinosorb M-active and Tinosorb S.

    PubMed

    Ashby, J; Tinwell, H; Plautz, J; Twomey, K; Lefevre, P A

    2001-12-01

    The presence of structurally diverse chemicals as contaminants in the environment has led to concerns regarding their possible endocrine disturbing effects. Recently, some ultraviolet absorbing components of sunscreen preparations have given positive responses in assays monitoring estrogen-like activity both in vitro and in vivo. Consequently, two recently developed sunscreen components, Tinosorb M-active and Tinosorb S, were evaluated using the in vitro estrogen and androgen receptor competitive binding assays. Neither compound gave a positive response in either of the assays, consistent with the large molecular dimensions of each chemical disfavoring binding to the hormone receptors. Both of the chemicals were inactive in immature rat uterotrophic assays conducted using the subcutaneous route of administration. It is concluded that neither of these agents possess intrinsic estrogenic/antiestrogenic or androgenic/antiandrogenic activity. The several positive control chemicals evaluated gave the expected positive responses in the assays used. PMID:11754532

  2. Influence of the membrane dipole potential on peptide binding to lipid bilayers

    PubMed Central

    Zhan, Huan; Lazaridis, Themis

    2011-01-01

    The implicit membrane model IMM1 is extended to include the membrane dipole potential and applied to molecular dynamics simulations of the helical peptides alamethicin, WALP23, influenza hemagglutinin fusion peptide, HIV fusion peptide, magainin, and the pre-sequence of cytochrome c oxidase subunit IV (p25). The results show that the orientation of the peptides in the membrane can be influenced by the dipole potential. The binding affinity of all peptides except for the hemagglutinin fusion peptide decreases upon increase of the dipole potential. The changes in both orientation and binding affinity are explained by the interaction of the dipole potential with the helix backbone dipole and ionic side-chains. In general, peptides that tend to insert the N-terminus in the membrane and/or have positively charged side chains will lose binding affinity upon increase of the dipole potential. PMID:22100997

  3. Membrane potential and surface potential in mitochondria: uptake and binding of lipophilic cations.

    PubMed

    Rottenberg, H

    1984-01-01

    The uptake and binding of the lipophilic cations ethidium+, tetraphenylphosphonium+ (TPP+), triphenylmethylphosphonium+ (TPMP+), and tetraphenylarsonium+ (TPA+) in rat liver mitochondria and submitochondrial particles were investigated. The effects of membrane potential, surface potentials and cation concentration on the uptake and binding were elucidated. The accumulation of these cations by mitochondria is described by an uptake and binding to the matrix face of the inner membrane in addition to the binding to the cytosolic face of the inner membrane. The apparent partition coefficients between the external medium and the cytosolic surface of the inner membrane (K'o) and the internal matrix volume and matrix face of the inner membrane (K'i) were determined and were utilized to estimate the membrane potential delta psi from the cation accumulation factor Rc according to the relation delta psi = RT/ZF ln [(RcVo - K'o)/(Vi + K'i)] where Vo and Vi are the volume of the external medium and the mitochondrial matrix, respectively, and Rc is the ratio of the cation content of the mitochondria and the medium. The values of delta psi estimated from this equation are in remarkably good agreement with those estimated from the distribution of 86Rb in the presence of valinomycin. The results are discussed in relation to studies in which the membrane potential in mitochondria and bacterial cells was estimated from the distribution of lipophilic cations. PMID:6492133

  4. Modelling Ser129 Phosphorylation Inhibits Membrane Binding of Pore-Forming Alpha-Synuclein Oligomers

    PubMed Central

    Nübling, Georg Sebastian; Levin, Johannes; Bader, Benedikt; Lorenzl, Stefan; Hillmer, Andreas; Högen, Tobias; Kamp, Frits; Giese, Armin

    2014-01-01

    Background In several neurodegenerative diseases, hyperphosphorylation at position Ser129 is found in fibrillar deposits of alpha-synuclein (asyn), implying a pathophysiological role of asyn phosphorylation in neurodegeneration. However, recent animal models applying asyn phosphorylation mimics demonstrated a protective effect of phosphorylation. Since metal-ion induced asyn oligomers were identified as a potential neurotoxic aggregate species with membrane pore-forming abilities, the current study was undertaken to determine effects of asyn phosphorylation on oligomer membrane binding. Methods We investigated the influence of S129 phosphorylation on interactions of metal-ion induced asyn oligomers with small unilamellar lipid vesicles (SUV) composed of POPC and DPPC applying the phosphorylation mimic asyn129E. Confocal single-particle fluorescence techniques were used to monitor membrane binding at the single-particle level. Results Binding of asyn129E monomers to gel-state membranes (DPPC-SUV) is slightly reduced compared to wild-type asyn, while no interactions with membranes in the liquid-crystalline state (POPC-SUV) are seen for both asyn and asyn129E. Conversely, metal-ion induced oligomer formation is markedly increased in asyn129E. Surprisingly, membrane binding to POPC-SUV is nearly absent in Fe3+ induced asyn129E oligomers and markedly reduced in Al3+ induced oligomers. Conclusion The protective effect of pseudophosphorylation seen in animal models may be due to impeded oligomer membrane binding. Phosphorylation at Ser129 may thus have a protective effect against neurotoxic asyn oligomers by preventing oligomer membrane binding and disruption of the cellular electrophysiological equilibrium. Importantly, these findings put a new complexion on experimental pharmaceutical interventions against POLO-2 kinase. PMID:24911099

  5. Genetic basis of endocrine disease 4: The spectrum of mutations in the androgen receptor gene that causes androgen resistance

    SciTech Connect

    McPhaul, M.J.; Marcelli, M.; Zoppi, S.; Griffin, J.E.; Wilson, J.D. )

    1993-01-01

    Mutations in the androgen receptor gene cause phenotypic abnormalities of male sexual development that range from a female phenotype (complete testicular feminization) to that of undervirilized or infertile men. Using the tools of molecular biology, the authors have analyzed androgen receptor gene mutations in 31 unrelated subjects with androgen resistance syndromes. Most of the defects are due to nucleotide changes that cause premature termination codons or single amino acid substitutions within the open reading frame encoding the androgen receptor, and the majority of these substitutions are localized in three regions of the androgen receptor: the DNA-binding domain and two segments of the androgen-binding domain. Less frequently, partial or complete gene deletions have been identified. Functional studies and immunoblot assays of the androgen receptors in patients with androgen resistance indicate that in most cases the phenotypic abnormalities are the result of impairment of receptor function or decreases in receptor abundance or both. 34 refs., 2 figs.

  6. Reconstitution of high-affinity opioid agonist binding in brain membranes

    SciTech Connect

    Remmers, A.E.; Medzihradsky, F. )

    1991-03-15

    In synaptosomal membranes from rat brain cortex, the {mu} selective agonist ({sup 3}H)dihydromorphine in the absence of sodium, and the nonselective antagonist ({sup 3}H)naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with K{sub d} values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 {mu}M guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP({gamma}-{sup 35}S) binding sites by 90% and low K{sub m}, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP({gamma}S) was diminished. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low K{sub m} GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor - guanine nucleotide binding protein complex.

  7. 2-Hydroxy Fatty Acid Enantiomers of Gb3 Impact Shiga Toxin Binding and Membrane Organization.

    PubMed

    Schütte, Ole M; Patalag, Lukas J; Weber, Lucas M C; Ries, Annika; Römer, Winfried; Werz, Daniel B; Steinem, Claudia

    2015-06-16

    Shiga toxin subunit B (STxB) binding to its cellular receptor Gb3 leads to the formation of protein-lipid clusters and bending of the membrane. A newly developed synthetic route allowed synthesizing the biologically most relevant Gb3-C24:1 2OH species with both, the natural (Gb3-R) as well as the unnatural (Gb3-S) configuration of the 2OH group. The derivatives bind STxB with identical nanomolar affinity, while the propensity to induce membrane tubules in giant unilamellar vesicles is more pronounced for Gb3-S. Fluorescence and atomic force microscopy images of phase-separated supported membranes revealed differences in the lateral organization of the protein on the membrane. Gb3-R favorably induces large and tightly packed protein clusters, while a lower protein density is found on Gb3-S doped membranes. PMID:26083916

  8. Androgen receptor genomic regulation

    PubMed Central

    Jin, Hong-Jian; Kim, Jung

    2013-01-01

    The transcriptional activity of the androgen receptor (AR) is not only critical for the normal development and function of the prostate but also pivotal to the onset and progression of prostate cancer (PCa). The studies of AR transcriptional regulation were previously limited to a handful of AR-target genes. Owing to the development of various high-throughput genomic technologies, significant advances have been made in recent years. Here we discuss the discoveries of genome-wide androgen-regulated genes in PCa cell lines, animal models and tissues using expression microarray and sequencing, the mapping of genomic landscapes of AR using Combining Chromatin Immunoprecipitation (ChIP)-on-chip and ChIP-seq assays, the interplay of transcriptional cofactors in defining AR binding profiles, and the genomic regulation and AR reprogramming in advanced PCa. PMID:25237629

  9. Increased expression of the androgen receptor with p300 and interleukin-6 coactivators compensate for oligonucleotide suppression of bcl-2: no increased CREB binding protein or interleukin-4 expression

    PubMed Central

    Hollowell, Courtney M.P.; Guinan, Patrick

    2013-01-01

    Background: Antisense oligonucleotides (oligos) have been employed against in vivo and in vitro prostate cancer models targeting growth regulatory proteins. While most oligos have targeted growth factors or their receptors, others have been directed against inhibitors of apoptosis and mediators of androgen action. We previously evaluated a set of oligos which targeted and comparably suppressed the expression of the apoptosis inhibitor protein bcl-2. LNCaP cells adapted to this restoration of apoptosis with suppression of caspase 3 (an apoptosis promoter) and an enhanced expression of the androgen receptor (AR), suggesting an increased sensitivity to androgens. Methods and results: In a continuation of this study, we evaluated the expression of AR coactivators p300, its homolog CREB binding protein (CREBBP) and cytokines interleukin (IL)-4 and IL-6, finding p300 and IL-6 similarly enhanced. Conclusions: LNCaP cells are hormone sensitive and untreated cells express minimal p300 activity. Therefore, the enhanced expression which followed oligo treatment makes its induction more impressive and implies a pattern of gene expression more associated with later stage (androgen insensitive) disease. This suggests that oligo treatment directed against bcl-2 can be evaded through compensatory changes in AR expression and some coactivators, promoting tumor growth, and may promote transformation of the tumor to a more aggressive phenotype. PMID:23554843

  10. Specific strychnine binding sites on acrosome-associated membranes of golden hamster spermatozoa.

    PubMed

    Llanos, Miguel N; Ronco, Ana M; Aguirre, María C

    2003-06-27

    This study demonstrates for the first time, that membrane vesicles originated from the hamster sperm head after the occurrence of the acrosome reaction, possess specific strychnine binding sites. [3H]Strychnine binding was saturable and reversible, being displaced by unlabeled strychnine (IC(50)=26.7+/-2.3 microM). Kinetic analysis revealed one binding site with K(d)=120nM and B(max)=142fmol/10(6) spermatozoa. Glycine receptor agonists beta-alanine and taurine inhibited strychnine binding by 20-30%. Surprisingly, glycine stimulated binding by about 40-50%. Results obtained in this study strongly suggest the presence of glycine receptors-with distinctive kinetic properties on the periacrosomal plasma membrane of hamster spermatozoa. Localization of this receptor fits well with its previously proposed role in acrosomal exocytosis during mammalian fertilization. PMID:12804573

  11. Disentangling Viral Membrane Fusion from Receptor Binding Using Synthetic DNA-Lipid Conjugates.

    PubMed

    Rawle, Robert J; Boxer, Steven G; Kasson, Peter M

    2016-07-12

    Enveloped viruses must bind to a receptor on the host membrane to initiate infection. Membrane fusion is subsequently initiated by a conformational change in the viral fusion protein, triggered by receptor binding, an environmental change, or both. Here, we present a strategy to disentangle the two processes of receptor binding and fusion using synthetic DNA-lipid conjugates to bind enveloped viruses to target membranes in the absence of receptor. This permits direct testing of whether receptor engagement affects the fusion mechanism as well as a comparison of fusion behavior across viruses with different receptor binding specificities. We demonstrate this approach by binding X-31 influenza virus to target vesicles and measuring the rates of individual pH-triggered lipid mixing events using fluorescence microscopy. Influenza lipid mixing kinetics are found to be independent of receptor binding, supporting the common yet previously unproven assumption that receptor binding does not produce any clustering or spatial rearrangement of viral hemagglutinin, which affects the rate-limiting step of pH-triggered fusion. This DNA-lipid tethering strategy should also allow the study of viruses where challenging receptor reconstitution has previously prevented single-virus fusion experiments. PMID:27410740

  12. Evaluation of androgen receptor and GATA binding protein 3 as immunohistochemical markers in the diagnosis of metastatic breast carcinoma to the lung.

    PubMed

    Hattori, Yukinori; Yoshida, Akihiko; Yoshida, Masayuki; Takahashi, Masahide; Tsuta, Koji

    2015-06-01

    Differentiating metastatic breast carcinoma in the lungs from primary lung tumors and mesotheliomas is important for determining prognosis and treatment. We evaluated novel breast specific markers, androgen receptor (AR) and GATA binding protein 3 (GATA3) immunohistostaining, for this differential, and compare to other traditional markers. The specimens comprised 33 metastatic breast carcinomas to the lung, 566 primary lung tumors (170 adenocarcinomas, 157 squamous cell carcinomas, 31 pleomorphic carcinomas, 115 large cell neuroendocrine carcinomas, 43 small cell carcinomas, and 49 typical carcinoids) and 42 malignant mesotheliomas. They were analyzed by immunohistochemistry using antibodies to AR, GATA3, estrogen receptor (ER), progesterone receptor (PgR), mammaglobin, gross cystic disease fluid protein-15 (GCDFP-15). Of the metastatic breast carcinomas, immunohistostaining of AR, GATA3, ER, PgR, mammaglobin, GCDFP-15 were positive in 27 cases (81.8%), 24 cases (72.7%), 26 cases (78.8%), 13 cases (39.4%), 12 cases (36.4%), 9 cases (27.3%), respectively. Of primary lung tumors and mesotheliomas, staining of AR, GATA3, ER, PgR, mammaglobin, GCDFP-15 were positive in 18 cases (3%), 3 cases (0.5%), 4 cases (0.7%), 2 cases (0.3%), 0 case (0%), 2 cases (0.3%), respectively. Immunohistochemistry of AR and GATA3 are reliable for differentiating metastatic breast carcinoma from primary lung tumors and mesotheliomas. PMID:25727644

  13. Isolation and partial characterization of a fatty acid binding protein in rat liver plasma membranes.

    PubMed Central

    Stremmel, W; Strohmeyer, G; Borchard, F; Kochwa, S; Berk, P D

    1985-01-01

    When [14C]oleate-bovine serum albumin complexes were incubated in vitro with rat liver plasma membranes (LPM), specific, saturable binding of oleate to the membranes was observed. Maximal heat-sensitive (i.e., specific) binding was 3.2 nmol/mg of membrane protein. Oleate-agarose affinity chromatography of Triton X-100-solubilized LPM was used to isolate a single 40-kDa protein with high affinity for oleate. On gel filtration, the protein comigrated with various fatty acids but not with [14C]bilirubin, [35S]sulfobromophthalein, [14C]taurocholate, [14C]phosphatidylcholine, or [14C]cholesteryloleate. A rabbit antibody to this membrane fatty acid-binding protein gave a single precipitin line with the antigen but no reactivity with concentrated cytosolic proteins, LPM bilirubin/sulfobromophthalein-binding protein, or rat albumin or other rat plasma proteins. The antibody selectively inhibited heat-sensitive binding of [14C]oleate to LPM. Immunofluorescence studies localized the antigen in liver-cell plasma membranes as well as in other major sites of fatty acid transport. These data are compatible with the hypothesis that this protein may act as a receptor in a hepatocellular uptake mechanism for fatty acids. Images PMID:3881757

  14. Identification of Novel Membrane-binding Domains in Multiple Yeast Cdc42 Effectors

    PubMed Central

    Takahashi, Satoe

    2007-01-01

    The Rho-type GTPase Cdc42 is a central regulator of eukaryotic cell polarity and signal transduction. In budding yeast, Cdc42 regulates polarity and mitogen-activated protein (MAP) kinase signaling in part through the PAK-family kinase Ste20. Activation of Ste20 requires a Cdc42/Rac interactive binding (CRIB) domain, which mediates its recruitment to membrane-associated Cdc42. Here, we identify a separate domain in Ste20 that interacts directly with membrane phospholipids and is critical for its function. This short region, termed the basic-rich (BR) domain, can target green fluorescent protein to the plasma membrane in vivo and binds PIP2-containing liposomes in vitro. Mutation of basic or hydrophobic residues in the BR domain abolishes polarized localization of Ste20 and its function in both MAP kinase–dependent and independent pathways. Thus, Cdc42 binding is required but is insufficient; instead, direct membrane binding by Ste20 is also required. Nevertheless, phospholipid specificity is not essential in vivo, because the BR domain can be replaced with several heterologous lipid-binding domains of varying lipid preferences. We also identify functionally important BR domains in two other yeast Cdc42 effectors, Gic1 and Gic2, suggesting that cooperation between protein–protein and protein–membrane interactions is a prevalent mechanism during Cdc42-regulated signaling and perhaps for other dynamic localization events at the cell cortex. PMID:17914055

  15. Multiple sup 3 H-oxytocin binding sites in rat myometrial plasma membranes

    SciTech Connect

    Crankshaw, D.; Gaspar, V.; Pliska, V. )

    1990-01-01

    The affinity spectrum method has been used to analyse binding isotherms for {sup 3}H-oxytocin to rat myometrial plasma membranes. Three populations of binding sites with dissociation constants (Kd) of 0.6-1.5 x 10(-9), 0.4-1.0 x 10(-7) and 7 x 10(-6) mol/l were identified and their existence verified by cluster analysis based on similarities between Kd, binding capacity and Hill coefficient. When experimental values were compared to theoretical curves constructed using the estimated binding parameters, good fits were obtained. Binding parameters obtained by this method were not influenced by the presence of GTP gamma S (guanosine-5'-O-3-thiotriphosphate) in the incubation medium. The binding parameters agree reasonably well with those found in uterine cells, they support the existence of a medium affinity site and may allow for an explanation of some of the discrepancies between binding and response in this system.

  16. Alternatively Spliced Androgen Receptor Variants

    PubMed Central

    Dehm, Scott M.; Tindall, Donald J.

    2011-01-01

    Alternative splicing is an important mechanism for increasing functional diversity from a limited set of genes. De-regulation of this process is common in diverse pathologic conditions. The androgen receptor (AR) is a steroid receptor transcription factor with functions critical for normal male development as well as the growth and survival of normal and cancerous prostate tissue. Studies of AR function in androgen insensitivity syndrome (AIS) and prostate cancer (PCa) have demonstrated loss-of-function AR alterations in AIS, and gain-of-function AR alterations in PCa. Over the past two decades, AR gene alterations have been identified in various individuals with AIS, which disrupt normal AR splicing patterns and yield dysfunctional AR protein variants. More recently, altered AR splicing patterns have been identified as a mechanism of PCa progression and resistance to androgen-depletion therapy. Several studies have described the synthesis of alternatively spliced transcripts encoding truncated AR isoforms that lack the ligand-binding domain, which is the ultimate target of androgen depletion. Many of these truncated AR isoforms function as constitutively active, ligand-independent transcription factors that can support androgen-independent expression of AR target genes, as well as the androgen-independent growth of PCa cells. In this review, we will summarize the various alternatively spliced AR variants that have been discovered, with a focus on their role and origin in the pathologic conditions of AIS and PCa. PMID:21778211

  17. Regulation of K-Ras4B Membrane Binding by Calmodulin.

    PubMed

    Sperlich, Benjamin; Kapoor, Shobhna; Waldmann, Herbert; Winter, Roland; Weise, Katrin

    2016-07-12

    K-Ras4B is a membrane-bound small GTPase with a prominent role in cancer development. It contains a polybasic farnesylated C-terminus that is required for the correct localization and clustering of K-Ras4B in distinct membrane domains. PDEδ and the Ca(2+)-binding protein calmodulin (CaM) are known to function as potential binding partners for farnesylated Ras proteins. However, they differ in the number of interaction sites with K-Ras4B, leading to different modes of interaction, and thus affect the subcellular distribution of K-Ras4B in different ways. Although it is clear that Ca(2+)-bound CaM can play a role in the dynamic spatial cycle of K-Ras4B in the cell, the exact molecular mechanism is only partially understood. In this biophysical study, we investigated the effect of Ca(2+)/CaM on the interaction of GDP- and GTP-loaded K-Ras4B with heterogeneous model biomembranes by using a combination of different spectroscopic and imaging techniques. The results show that Ca(2+)/CaM is able to extract K-Ras4B from negatively charged membranes in a nucleotide-independent manner. Moreover, the data demonstrate that the complex of Ca(2+)/CaM and K-Ras4B is stable in the presence of anionic membranes and shows no membrane binding. Finally, the influence of Ca(2+)/CaM on the interaction of K-Ras4B with membranes is compared with that of PDEδ, which was investigated in a previous study. Although both CaM and PDEδ exhibit a hydrophobic binding pocket for farnesyl, they have different effects on membrane binding of K-Ras4B and hence should be capable of regulating K-Ras4B plasma membrane localization in the cell. PMID:27410739

  18. Cross-linking of atrial natriuretic peptide to binding sites in rat olfactory bulb membranes

    SciTech Connect

    Wildey, G.M.; Glembotski, C.C.

    1986-12-01

    Binding sites for /sup 125/I-atrial natriuretic peptide (ANP)2 in rat olfactory bulb membranes have been studied using pharmacological and biochemical methods. Various unlabeled ANP-related peptides were tested for the ability to inhibit the binding of the radioligand in membrane binding assays. ANP(92-126) and ANP(99-126) were the most potent inhibitors tested, both exhibiting an IC50 value of 0.40 nM. ANP(103-126) and ANP(103-123) were 3 and 70 times less potent, respectively. ANP(111-126) was unable to inhibit the binding of the radioligand at a concentration of 1 microM. Several peptides unrelated to ANP were unable to inhibit the binding of the radioligand to rat olfactory bulb membranes. Membranes labeled with /sup 125/I-ANP were incubated with cross-linking agents and subjected to SDS-PAGE followed by autoradiography. A band possessing an apparent molecular mass of 116 kDa was identified. The labeling of this band was progressively decreased by increasing concentrations of unlabeled ANP(99-126) (IC50 = 0.6 nM) and by several other ANP-related peptides at nanomolar concentrations. For comparison purposes, ANP binding sites in rat aorta membranes were labeled with /sup 125/I-ANP and cross-linked using identical techniques. Three bands possessing molecular masses of 120, 72, and 62 kDa were identified. These results indicate that the ANP binding site in rat olfactory bulb membranes displays pharmacological and biochemical properties similar to peripheral ANP receptors.

  19. The membrane bound bacterial lipocalin Blc is a functional dimer with binding preference for lysophospholipids

    PubMed Central

    Campanacci, Valérie; Bishop, Russell E.; Blangy, Stéphanie; Tegoni, Mariella; Cambillau, Christian

    2016-01-01

    Lipocalins, a widespread multifunctional family of small proteins (15–25 kDa) have been first described in eukaryotes and more recently in Gram-negative bacteria. Bacterial lipocalins belonging to class I are outer membrane lipoproteins, among which Blc from E. coli is the better studied. Blc is expressed under conditions of starvation and high osmolarity, conditions known to exert stress on the cell envelope. The structure of Blc that we have previously solved (V. Campanacci, D. Nurizzo, S. Spinelli, C. Valencia, M. Tegoni, C. Cambillau, FEBS Lett. 562 (2004) 183–188.) suggested its possible role in binding fatty acids or phospholipids. Both physiological and structural data on Blc, therefore, point to a role in storage or transport of lipids necessary for membrane maintenance. In order to further document this hypothesis for Blc function, we have performed binding studies using fluorescence quenching experiments. Our results indicate that dimeric Blc binds fatty acids and phospholipids in a micromolar Kd range. The crystal structure of Blc with vaccenic acid, an unsaturated C18 fatty acid, reveals that the binding site spans across the Blc dimer, opposite to its membrane anchored face. An exposed unfilled pocket seemingly suited to bind a polar group attached to the fatty acid prompted us to investigate lyso-phospholipids, which were found to bind in a nanomolar Kd range. We discuss these findings in terms of a potential role for Blc in the metabolism of lysophospholipids generated in the bacterial outer membrane. PMID:16920109

  20. Identification of high affinity folate binding proteins in human erythrocyte membranes.

    PubMed

    Antony, A C; Kincade, R S; Verma, R S; Krishnan, S R

    1987-09-01

    Mature human erythrocyte membranes contained specific, high affinity (Kd 3.3 X 10(-11) M) folate binding moieties. Folate binding was pH, time- and temperature-dependent, saturable, and was much greater for pteroylmonoglutamate and 5-methyltetrahydrofolate than 5-formyltetrahydrofolate and amethopterin. On detergent solubilization of membranes, two peaks of specific folate binding with Mr greater than or equal to 200,000 and 160,000 were identified on Sephacryl S-200 gel filtration chromatography in Triton X-100, and this corresponded to two similar peaks of immunoprecipitated material when solubilized iodinated membranes were probed with anti-human placental folate receptor antiserum. Age-dependent separation of erythrocytes by Stractan density gradients revealed a sevenfold greater folate binding capacity in membranes purified from younger compared with aged erythrocytes. Since this difference was not reflected in proportionately higher immunoreactive folate binding protein, (as determined by a specific radioimmunoassay for these proteins), or differences in affinity in younger than aged cells, these findings indicate that erythrocyte folate binding proteins become progressively nonfunctional at the onset of red cell aging. PMID:3624486

  1. Tau Binds to Lipid Membrane Surfaces via Short Amphipathic Helices Located in Its Microtubule-Binding Repeats

    PubMed Central

    Georgieva, Elka R.; Xiao, Shifeng; Borbat, Peter P.; Freed, Jack H.; Eliezer, David

    2014-01-01

    Tau is a microtubule-associated protein that is genetically linked to dementia and linked to Alzheimer’s disease via its presence in intraneuronal neurofibrillary tangle deposits, where it takes the form of aggregated paired helical and straight filaments. Although the precise mechanisms by which tau contributes to neurodegeneration remain unclear, tau aggregation is commonly considered to be a critical component of tau-mediated pathogenicity. Nevertheless, the context in which tau aggregation begins in vivo is unknown. Tau is enriched in membrane-rich neuronal structures such as axons and growth cones, and can interact with membranes both via intermediary proteins and directly via its microtubule-binding domain (MBD). Membranes efficiently facilitate tau aggregation in vitro, and may therefore provide a physiologically relevant context for nucleating tau aggregation in vivo. Furthermore, tau-membrane interactions may potentially play a role in tau’s poorly understood normal physiological functions. Despite the potential importance of direct tau-membrane interactions for tau pathology and physiology, the structural mechanisms that underlie such interactions remain to be elucidated. Here, we employ electron spin resonance spectroscopy to investigate the secondary and long-range structural properties of the MBD of three-repeat tau isoforms when bound to lipid vesicles and membrane mimetics. We show that the membrane interactions of the tau MBD are mediated by short amphipathic helices formed within each of the MBD repeats in the membrane-bound state. To our knowledge, this is the first detailed elucidation of helical tau structure in the context of intact lipid bilayers. We further show, for the first time (to our knowledge), that these individual helical regions behave as independent membrane-binding sites linked by flexible connecting regions. These results represent the first (to our knowledge) detailed structural view of membrane-bound tau and provide

  2. Outer membrane protein DsrA is the major fibronectin-binding determinant of Haemophilus ducreyi.

    PubMed

    Leduc, Isabelle; White, C Dinitra; Nepluev, Igor; Throm, Robert E; Spinola, Stanley M; Elkins, Christopher

    2008-04-01

    The ability to bind extracellular matrix proteins is a critical virulence determinant for skin pathogens. Haemophilus ducreyi, the etiological agent of the genital ulcer disease chancroid, binds extracellular matrix components, including fibronectin (FN). We investigated H. ducreyi FN binding and report several important findings about this interaction. First, FN binding by H. ducreyi was greatly increased in bacteria grown on heme and almost completely inhibited by hemoglobin. Second, wild-type strain 35000HP bound significantly more FN than did a dsrA mutant in two different FN binding assays. Third, the expression of dsrA in the dsrA mutant restored FN binding and conferred the ability to bind FN to a non-FN-binding Haemophilus influenzae strain. Fourth, an anti-DsrA monoclonal antibody partially blocked FN binding by H. ducreyi. The hemoglobin receptor, the collagen-binding protein, the H. ducreyi lectin, the fine-tangle pili, and the outer membrane protein OmpA2 were not involved in H. ducreyi FN binding, since single mutants bound FN as well as the parent strain did. However, the major outer membrane protein may have a minor role in FN binding by H. ducreyi, since a double dsrA momp mutant bound less FN than did the single dsrA mutant. Finally, despite major sequence differences, DsrA proteins from both class I and class II H. ducreyi strains mediated FN and vitronectin binding. We concluded that DsrA is the major factor involved in FN binding by both classes of H. ducreyi strains. PMID:18212073

  3. Zinc Binding to MG53 Protein Facilitates Repair of Injury to Cell Membranes*

    PubMed Central

    Cai, Chuanxi; Lin, Peihui; Zhu, Hua; Ko, Jae-Kyun; Hwang, Moonsun; Tan, Tao; Pan, Zui; Korichneva, Irina; Ma, Jianjie

    2015-01-01

    Zinc is an essential trace element that participates in a wide range of biological functions, including wound healing. Although Zn2+ deficiency has been linked to compromised wound healing and tissue repair in human diseases, the molecular mechanisms underlying Zn2+-mediated tissue repair remain unknown. Our previous studies established that MG53, a TRIM (tripartite motif) family protein, is an essential component of the cell membrane repair machinery. Domain homology analysis revealed that MG53 contains two Zn2+-binding motifs. Here, we show that Zn2+ binding to MG53 is indispensable to assembly of the cell membrane repair machinery. Live cell imaging illustrated that Zn2+ entry from extracellular space is essential for translocation of MG53-containing vesicles to the acute membrane injury sites for formation of a repair patch. The effect of Zn2+ on membrane repair is abolished in mg53−/− muscle fibers, suggesting that MG53 functions as a potential target for Zn2+ during membrane repair. Mutagenesis studies suggested that both RING and B-box motifs of MG53 constitute Zn2+-binding domains that contribute to MG53-mediated membrane repair. Overall, this study establishes a base for Zn2+ interaction with MG53 in protection against injury to the cell membrane. PMID:25869134

  4. Curvature sensing MARCKS-ED peptides bind to membranes in a stereo-independent manner.

    PubMed

    Yan, Lei; de Jesus, Armando Jerome; Tamura, Ryo; Li, Victoria; Cheng, Kui; Yin, Hang

    2015-07-01

    Membrane curvature and lipid composition plays a critical role in interchanging of matter and energy in cells. Peptide curvature sensors are known to activate signaling pathways and promote molecular transport across cell membranes. Recently, the 25-mer MARCKS-ED peptide, which is derived from the effector domain of the myristoylated alanine-rich C kinase substrate protein, has been reported to selectively recognize highly curved membrane surfaces. Our previous studies indicated that the naturally occurring L-MARCKS-ED peptide could simultaneously detect both phosphatidylserine and curvature. Here, we demonstrate that D-MARCKS-ED, composed by unnatural D-amino acids, has the same activities as its enantiomer, L-MARCKS-ED, as a curvature and lipid sensor. An atomistic molecular dynamics simulation suggests that D-MARCKS-ED may change from linear to a boat conformation upon binding to the membrane. Comparable enhancement of fluorescence intensity was observed between D- and L-MARCKS-ED peptides, indicating similar binding affinities. Meanwhile, circular dichroism spectra of D- and L-MARCKS-ED are almost symmetrical both in the presence and absence of liposomes. These results suggest similar behavior of artificial D- and natural L-MARCKS-ED peptides when binding to curved membranes. Our studies may contribute to further understanding of how MARCKS-ED senses membrane curvature as well as provide a new direction to develop novel membrane curvature probes. PMID:25851418

  5. Zinc Binding to MG53 Protein Facilitates Repair of Injury to Cell Membranes.

    PubMed

    Cai, Chuanxi; Lin, Peihui; Zhu, Hua; Ko, Jae-Kyun; Hwang, Moonsun; Tan, Tao; Pan, Zui; Korichneva, Irina; Ma, Jianjie

    2015-05-29

    Zinc is an essential trace element that participates in a wide range of biological functions, including wound healing. Although Zn(2+) deficiency has been linked to compromised wound healing and tissue repair in human diseases, the molecular mechanisms underlying Zn(2+)-mediated tissue repair remain unknown. Our previous studies established that MG53, a TRIM (tripartite motif) family protein, is an essential component of the cell membrane repair machinery. Domain homology analysis revealed that MG53 contains two Zn(2+)-binding motifs. Here, we show that Zn(2+) binding to MG53 is indispensable to assembly of the cell membrane repair machinery. Live cell imaging illustrated that Zn(2+) entry from extracellular space is essential for translocation of MG53-containing vesicles to the acute membrane injury sites for formation of a repair patch. The effect of Zn(2+) on membrane repair is abolished in mg53(-/-) muscle fibers, suggesting that MG53 functions as a potential target for Zn(2+) during membrane repair. Mutagenesis studies suggested that both RING and B-box motifs of MG53 constitute Zn(2+)-binding domains that contribute to MG53-mediated membrane repair. Overall, this study establishes a base for Zn(2+) interaction with MG53 in protection against injury to the cell membrane. PMID:25869134

  6. (/sup 3/H)nitrendipine binding to adrenal capsular membranes

    SciTech Connect

    Finkel, M.S.; Aguilera, G.; Catt, K.J.; Keiser, H.R.

    1984-08-20

    The physiologic regulation of aldosterone secretion is dependent on extracellular calcium and appears to be mediated by increases in cytosolic free calcium concentration in the zona glomerulosa cell. A specific role for voltage-dependent calcium channels was suggested by previous studies with the calcium channel antagonist verapamil. The authors therefore studied the (/sup 3/H)nitrendipine calcium channel binding site in adrenal capsules. These studies revealed a single class of saturable, high affinity sites with K/sub D/ = .26 +/- .04 nM and B/sub max/ = 105 +/- 5.7 fmol/mg protein. Specific binding of (/sup 3/H)nitrendipine was inhibited by calcium channel antagonists with potencies nitrendipine = nifedipine >> verapamil, while diltiazem had no inhibitory effect. In the rat, binding sites for (/sup 3/H)nitrendipine were located in the adrenal capsule and medulla and were undetectable in the zona fasciculata. Physiologic studies with collagenase-dispersed adrenal glomerulosa cells demonstrated that nifedipine selectively inhibited angiotensin-II and potassium-stimulated steroidogenesis. These observations suggest both a pharmacologic and physiologic role for the nitrendipine binding site in aldosterone production. 17 references, 2 figures, 1 table.

  7. Selective modulation by membrane potential of desmethoxyverapamil binding to calcium channels in rat portal vein.

    PubMed

    Rakotoarisoa, L; Sayet, I; Mironneau, C; Mironneau, J

    1990-12-01

    (-)-[3H]Desmethoxyverapamil (D888) binds saturably to intact strips from rat portal vein bathed in physiological solution with a Kd value of 363 pM and a maximal binding capacity value of 15.6 fmol.mg-1 wet weight. Unlabeled dihydropyridines, phenylalkylamines and benzothiazepines inhibited (-)-[3H]D888 specific binding in a concentration-dependent manner. Scatchard analyses and dissociation kinetics of (-)-[3H]D888 binding revealed the existence of mutual allosteric interactions between (+)-isradipine, (+)-cis diltiazem and (-)-D888 binding sites in portal vein strips. When voltage-dependent Ca++ channels transported Ca++, Ba++, Sr++ or Na+ the binding capacity of (-)-[3H]D888 remained unchanged. In contrast, both depolarization (induced by elevation of external K+) and hyperpolarization (in the presence of cromakalim) induced a gradual decrease in (-)-D888 binding capacity. These observations suggest that membrane potential variation would change the conformational state of Ca++ channels, in such a way that it would be less favorable for access of (-)-[3H]D888 to the binding site. This would provide an experimental argument in favor of the "guarded receptor hypothesis" according to which membrane potential modulates ligand affinity by alteration of the amount of time during which the receptor binding site is available to (-)-[3H]D888. PMID:2175807

  8. Membrane Binding and Self-Association of the Epsin N-Terminal Homology Domain

    PubMed Central

    Lai, Chun-Liang; Jao, Christine C.; Lyman, Edward; Gallop, Jennifer L.; Peter, Brian J.; McMahon, Harvey T.; Langen, Ralf; Voth, Gregory A.

    2012-01-01

    Epsin possesses a conserved epsin N-terminal homology (ENTH) domain that acts as a phosphatidylinositol 4,5-bisphosphate‐lipid‐targeting and membrane‐curvature‐generating element. Upon binding phosphatidylinositol 4,5‐bisphosphate, the N-terminal helix (H0) of the ENTH domain becomes structured and aids in the aggregation of ENTH domains, which results in extensive membrane remodeling. In this article, atomistic and coarse-grained (CG) molecular dynamics (MD) simulations are used to investigate the structure and the stability of ENTH domain aggregates on lipid bilayers. EPR experiments are also reported for systems composed of different ENTH-bound membrane morphologies, including membrane vesicles as well as preformed membrane tubules. The EPR data are used to help develop a molecular model of ENTH domain aggregates on preformed lipid tubules that are then studied by CG MD simulation. The combined computational and experimental approach suggests that ENTH domains exist predominantly as monomers on vesiculated structures, while ENTH domains self-associate into dimeric structures and even higher‐order oligomers on the membrane tubes. The results emphasize that the arrangement of ENTH domain aggregates depends strongly on whether the local membrane curvature is isotropic or anisotropic. The molecular mechanism of ENTH‐domain-induced membrane vesiculation and tubulation and the implications of the epsin's role in clathrin-mediated endocytosis resulting from the interplay between ENTH domain membrane binding and ENTH domain self-association are also discussed. PMID:22922484

  9. Location-specific nanoplasmonic sensing of biomolecular binding to lipid membranes with negative curvature

    NASA Astrophysics Data System (ADS)

    Junesch, Juliane; Emilsson, Gustav; Xiong, Kunli; Kumar, Shailabh; Sannomiya, Takumi; Pace, Hudson; Vörös, Janos; Oh, Sang-Hyun; Bally, Marta; Dahlin, Andreas B.

    2015-09-01

    The biochemical processes of cell membranes are sensitive to the geometry of the lipid bilayer. We show how plasmonic ``nanowells'' provide label-free real-time analysis of molecules on membranes with detection of preferential binding at negative curvature. It is demonstrated that norovirus accumulate in invaginations due to multivalent interactions with glycosphingolipids.The biochemical processes of cell membranes are sensitive to the geometry of the lipid bilayer. We show how plasmonic ``nanowells'' provide label-free real-time analysis of molecules on membranes with detection of preferential binding at negative curvature. It is demonstrated that norovirus accumulate in invaginations due to multivalent interactions with glycosphingolipids. Electronic supplementary information (ESI) available: Additional plasmonic sensing results, numerical electromagnetic simulations, quartz crystal microbalance data, fluorescence recovery after photobleaching, additional electron microscopy images, experimental methodology and materials used. See DOI: 10.1039/c5nr04208a

  10. Rapid preparation of plasma membranes from avian lymphoid cells and fibroblasts for virus binding studies.

    PubMed

    Nieper, H; Müller, H

    1998-06-01

    A simple and rapid protocol for the preparation of plasma membranes from chicken embryo fibroblasts and chicken lymphoid cells was developed. Characterization of the preparations by morphological, biochemical and serological methods indicated the specific enrichment of the plasma membranes as well as cell surface proteins. Binding of infectious bursal disease virus (IBDV) particles was demonstrated after immobilization of the plasma membranes, and cell type-specific differences were observed. Although the results of these studies reflect the interaction between IBDV and isolated cells only partially, the advantages of these plasma membrane preparations, the specific enrichment of cell surface proteins, their constant quality and the possibility to store aliquots over several months, make them a useful tool for virus binding studies with avian cells. PMID:9694323

  11. Roles played by acidic lipids in HIV-1 Gag membrane binding

    PubMed Central

    Olety, Balaji; Ono, Akira

    2014-01-01

    The MA domain mediates plasma membrane (PM) targeting of HIV-1 Gag, leading to particle assembly at the PM. The interaction between MA and acidic phospholipids, in addition to N-terminal myristoyl moiety, promotes Gag binding to lipid membranes. Among acidic phospholipids, PI(4,5)P2, a PM-specific phosphoinositide, is essential for proper HIV-1 Gag localization to the PM and efficient virus particle production. Recent studies further revealed that MA-bound RNA negatively regulates HIV-1 Gag membrane binding and that PI(4,5)P2 is necessary to overcome this RNA-imposed block. In this review, we will summarize the current understanding of Gag-membrane interactions and discuss potential roles played by acidic phospholipids. PMID:24998886

  12. Prediction of lipid-binding regions in cytoplasmic and extracellular loops of membrane proteins as exemplified by protein translocation membrane proteins.

    PubMed

    Keller, Rob C A

    2013-01-01

    The presence of possible lipid-binding regions in the cytoplasmic or extracellular loops of membrane proteins with an emphasis on protein translocation membrane proteins was investigated in this study using bioinformatics. Recent developments in approaches recognizing lipid-binding regions in proteins were found to be promising. In this study a total bioinformatics approach specialized in identifying lipid-binding helical regions in proteins was explored. Two features of the protein translocation membrane proteins, the position of the transmembrane regions and the identification of additional lipid-binding regions, were analyzed. A number of well-studied protein translocation membrane protein structures were checked in order to demonstrate the predictive value of the bioinformatics approach. Furthermore, the results demonstrated that lipid-binding regions in the cytoplasmic and extracellular loops in protein translocation membrane proteins can be predicted, and it is proposed that the interaction of these regions with phospholipids is important for proper functioning during protein translocation. PMID:22961045

  13. α-Bungarotoxin Binding to Acetylcholine Receptor Membranes Studied by Low Angle X-Ray Diffraction

    PubMed Central

    Young, Howard S.; Herbette, Leo G.; Skita, Victor

    2003-01-01

    The nicotinic acetylcholine receptor (nAChR) carries two binding sites for snake venom neurotoxins. α-Bungarotoxin from the Southeast Asian banded krait, Bungarus multicinctus, is a long neurotoxin which competitively blocks the nAChR at the acetylcholine binding sites in a relatively irreversible manner. Low angle x-ray diffraction was used to generate electron density profile structures at 14-Å resolution for Torpedo californica nAChR membranes in the absence and presence of α-bungarotoxin. Analysis of the lamellar diffraction data indicated a 452-Å lattice spacing between stacked nAChR membrane pairs. In the presence of α-bungarotoxin, the quality of the diffraction data and the lamellar lattice spacing were unchanged. In the plane of the membrane, the nAChRs packed together with a nearest neighbor distance of 80 Å, and this distance increased to 85 Å in the presence of toxin. Electron density profile structures were calculated in the absence and presence of α-bungarotoxin, revealing a location for the toxin binding sites. In native, fully-hydrated nAChR membranes, α-bungarotoxin binds to the nAChR outer vestibule and contacts the surface of the membrane bilayer. PMID:12885641

  14. External location of sites on pig erythrocyte membranes that bind nitrobenzylthioinosine

    SciTech Connect

    Agbanyo, F.R.; Cass, C.E.; Paterson, A.R.

    1988-03-01

    Nucleoside transport in erythrocytes of various species is inhibited by the binding of nitrobenzylthioinosine (NBMPR) to high affinity sites associated with nucleoside transport elements of the plasma membrane. The present study examined binding of (/sup 3/H)NBMPR to unsealed ghosts and to sealed right-side-out vesicles (ROVs) and inside-out vesicles (IOVs) prepared from pig erythrocytes. Kd values for NBMPR dissociation from the ligand-site complex in unsealed ghosts, ROVs and IOVs were similar (1.6-2.4 nM), and Bmax values (mean +/- SD) were, respectively, 22.2 +/- 5.5, 25.8 +/- 6.4, and 37.3 +/- 4.0 molecules/fg of protein, reflecting differences in the protein content of the membrane preparations. When temperatures were decreased from 22 degrees to 4 degrees, NBMPR binding to erythrocyte membrane preparations was reduced in IOVs relative to that in unsealed ghosts and ROVs. At 22 degrees, the association of NBMPR molecules with IOVs was slower than with ROVs and unsealed ghosts, differences that were virtually eliminated by permeabilization of the membrane preparations with saponin. Thus, the binding sites were more accessible to external NBMPR in sealed ROVs and unsealed ghosts than in sealed IOVs, indicating that the NBMPR sites are located on the extracellular aspect of the membrane.

  15. Surface area of lipid membranes regulates the DNA-binding capacity of cationic liposomes

    NASA Astrophysics Data System (ADS)

    Marchini, Cristina; Montani, Maura; Amici, Augusto; Pozzi, Daniela; Caminiti, Ruggero; Caracciolo, Giulio

    2009-01-01

    We have applied electrophoresis on agarose gels to investigate the DNA-binding capacity of cationic liposomes made of cationic DC-cholesterol and neutral dioleoylphosphatidylethanolamine as a function of membrane charge density and cationic lipid/DNA charge ratio. While each cationic liposome formulation exhibits a distinctive DNA-protection ability, here we show that such a capacity is universally regulated by surface area of lipid membranes available for binding in an aspecific manner. The relevance of DNA protection for gene transfection is also discussed.

  16. Human Rhinovirus Subviral A Particle Binds to Lipid Membranes over a Twofold Axis of Icosahedral Symmetry

    PubMed Central

    Kumar, Mohit

    2013-01-01

    Minor group human rhinoviruses bind low-density lipoprotein (LDL) receptors for endocytosis. Once they are inside endosomes, the acidic pH triggers their dissociation from the receptors and conversion into hydrophobic subviral A particles; these attach to the membrane and transfer their single-strand, positive-sense RNA genome into the cytosol. Here, we allowed human rhinovirus 2 (HRV2) A particles, produced in vitro by incubation at pH 5.4, to attach to liposomes; cryo-electron microscopy 3-dimensional single-particle image reconstruction revealed that they bind to the membrane around a 2-fold icosahedral symmetry axis. PMID:23946453

  17. A 138-kDa glycoprotein from Dictyostelium membranes with folate deaminase and folate binding activity.

    PubMed

    Greiner, R A; Jacobs-Krahnen, D; Mutzel, R; Malchow, D; Wurster, B

    1992-03-15

    A 138-kDa glycoprotein comprising folate deaminase activity was purified to apparent homogeneity from membranes of Dictyostelium discoideum. Deaminase activity could be effectively inhibited by p-chloromercuriphenylsulfonate. This treatment protected folate from deamination and thus allowed investigation of folate binding to deaminase fractions. Two types of folate binding sites, differing in affinity and specificity, were detected on the folate deaminase glycoprotein. One type displays high affinity and binds folate stronger than N10-methylfolate. This binding site appears to be identical with the catalytic site of folate deaminase. The other type of binding site shows lower affinity but prefers N10-methylfolate relative to folate. A similar preference for N10-methylfolate was observed in chemotaxis tests pointing to the possibility that the second type of binding site is involved in chemotactic perception of folate compounds. Folate perception and deamination could thus be performed by activities residing on the same polypeptide. PMID:1544893

  18. Characterization of Naphthaleneacetic Acid Binding to Receptor Sites on Cellular Membranes of Maize Coleoptile Tissue 1

    PubMed Central

    Ray, Peter M.; Dohrmann, Ulrike; Hertel, Rainer

    1977-01-01

    Characteristics of and optimum conditions for saturable (“specific”) binding of [14C]naphthaleneacetic acid to sites located on membranous particles from maize (Zea mays L.) coleoptiles are described. Most, if not all, of the specific binding appears to be due to a single kinetic class of binding sites having a KD of 5 to 7 × 10−7m for naphthalene-1-acetic acid (NAA). Binding of NAA is insensitive to high monovalent salt concentrations, indicating that binding is not primarily ionic. However, specific binding is inhibited by Mg2+ or Ca2+ above 5 mm. Specific binding is improved by organic acids, especially citrate. Binding is heat-labile and is sensitive to agents that act either on proteins or on lipids. Specific binding is reversibly inactivated by reducing agents such as dithioerythritol; a reducible group, possibly a disulfide group, may be located at the binding site and required for its function. The affinity of the specific binding sites for auxins is modified by an unidentified dialyzable, heat-stable, apparently amphoteric, organic factor (“supernatant factor”) found in maize tissue. PMID:16659851

  19. Phosphatidylethanolamine Binding Is a Conserved Feature of Cyclotide-Membrane Interactions*

    PubMed Central

    Henriques, Sónia Troeira; Huang, Yen-Hua; Castanho, Miguel A. R. B.; Bagatolli, Luis A.; Sonza, Secondo; Tachedjian, Gilda; Daly, Norelle L.; Craik, David J.

    2012-01-01

    Cyclotides are bioactive cyclic peptides isolated from plants that are characterized by a topologically complex structure and exceptional resistance to enzymatic or thermal degradation. With their sequence diversity, ultra-stable core structural motif, and range of bioactivities, cyclotides are regarded as a combinatorial peptide template with potential applications in drug design. The mode of action of cyclotides remains elusive, but all reported biological activities are consistent with a mechanism involving membrane interactions. In this study, a diverse set of cyclotides from the two major subfamilies, Möbius and bracelet, and an all-d mirror image form, were examined to determine their mode of action. Their lipid selectivity and membrane affinity were determined, as were their toxicities against a range of targets (red blood cells, bacteria, and HIV particles). Although they had different membrane-binding affinities, all of the tested cyclotides targeted membranes through binding to phospholipids containing phosphatidylethanolamine headgroups. Furthermore, the biological potency of the tested cyclotides broadly correlated with their ability to target and disrupt cell membranes. The finding that a broad range of cyclotides target a specific lipid suggests their categorization as a new lipid-binding protein family. Knowledge of their membrane specificity has the potential to assist in the design of novel drugs based on the cyclotide framework, perhaps allowing the targeting of peptide drugs to specific cell types. PMID:22854971

  20. Binding of small basic peptides to membranes containing acidic lipids: theoretical models and experimental results.

    PubMed Central

    Ben-Tal, N; Honig, B; Peitzsch, R M; Denisov, G; McLaughlin, S

    1996-01-01

    We measured directly the binding of Lys3, Lys5, and Lys7 to vesicles containing acidic phospholipids. When the vesicles contain 33% acidic lipids and the aqueous solution contains 100 mM monovalent salt, the standard Gibbs free energy for the binding of these peptides is 3, 5, and 7 kcal/mol, respectively. The binding energies decrease as the mol% of acidic lipids in the membrane decreases and/or as the salt concentration increases. Several lines of evidence suggest that these hydrophilic peptides do not penetrate the polar headgroup region of the membrane and that the binding is mainly due to electrostatic interactions. To calculate the binding energies from classical electrostatics, we applied the nonlinear Poisson-Boltzmann equation to atomic models of the phospholipid bilayers and the basic peptides in aqueous solution. The electrostatic free energy of interaction, which arises from both a long-range coulombic attraction between the positively charged peptide and the negatively charged lipid bilayer, and a short-range Born or image charge repulsion, is a minimum when approximately 2.5 A (i.e., one layer of water) exists between the van der Waals surfaces of the peptide and the lipid bilayer. The calculated molar association constants, K, agree well with the measured values: K is typically about 10-fold smaller than the experimental value (i.e., a difference of about 1.5 kcal/mol in the free energy of binding). The predicted dependence of K (or the binding free energies) on the ionic strength of the solution, the mol% of acidic lipids in the membrane, and the number of basic residues in the peptide agree very well with the experimental measurements. These calculations are relevant to the membrane binding of a number of important proteins that contain clusters of basic residues. Images FIGURE 2 FIGURE 3 PMID:8842196

  1. Role of calcium in membrane interactions by PI(4,5)P₂-binding proteins.

    PubMed

    Monteiro, Marina E; Sarmento, Maria J; Fernandes, Fábio

    2014-10-01

    Ca²⁺ and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] are key agents in membrane-associated signalling events. Their temporal and spatial regulation is crucial for activation or recruitment of proteins in the plasma membrane. In fact, the interaction of several signalling proteins with PI(4,5)P₂ has been shown to be tightly regulated and dependent on the presence of Ca²⁺, with co-operative binding in some cases. In these proteins, PI(4,5)P₂ and Ca²⁺ binding typically occurs at different binding sites. In addition, several PI(4,5)P₂-binding proteins are known targets of calmodulin (CaM), which, depending on the presence of calcium, can compete with PI(4,5)P₂ for protein interaction, translating Ca²⁺ transient microdomains into variations of PI(4,5)P₂ lateral organization in time and space. The present review highlights different examples of calcium-dependent PI(4,5)P₂-binding proteins and discusses the possible impact of this dual regulation on fine-tuning of protein activity by triggering target membrane binding in the presence of subtle changes in the levels of calcium or PI(4,5)P₂. PMID:25233429

  2. Specific binding of lactoferrin to brush-border membrane: Ontogeny and effect of glycan chain

    SciTech Connect

    Davidson, L.A.; Loennerdal, B. )

    1988-04-01

    Bioavailability of iron from human milk is exceptionally high. It has been suggested that lactoferrin, the major iron-binding protein in human milk, may participate in this high iron bioavailability from milk. The authors examined the interaction of lactoferrin with the intestinal brush-border membrane using the rhesus monkeys as a model. Brush-border membrane vesicles were prepared from monkeys of various ages. Binding studies with {sup 59}Fe-labeled human and monkey lactoferrin were performed to examine interaction of lactoferrin with the brush-border membrane. Specific saturable binding of lactoferrin was found at all ages studied. The dissociation constant for lactoferrin-receptor binding was 9 {times} 10{sup {minus}6} M. In contrast, no binding of serum transferrin or bovine lactoferrin occurred. Removal of fucose from the lactoferrin glycans resulted in a significant decrease in binding. It was concluded that lactoferrin in milk may function in the process of iron absorption through interaction with a small intestinal receptor and that fucosylated glycans on the carbohydrate chain of lactoferrin are necessary for receptor recognition.

  3. Molecular pathways: targeting resistance in the androgen receptor for therapeutic benefit.

    PubMed

    Mostaghel, Elahe A; Plymate, Stephen R; Montgomery, Bruce

    2014-02-15

    Androgen receptor signaling is critical in the development and progression of prostate cancer, leading to intensive efforts to elucidate all potential points of inflection for therapeutic intervention. These efforts have revealed new mechanisms of resistance and raise the possibility that known mechanisms may become even more relevant in the context of effective androgen receptor suppression. These mechanisms include tumoral appropriation of alternative androgen sources, alterations in androgen receptor expression, androgen receptor mutations, truncated androgen receptor variants, alterations and cross-talk in recruitment of cofactors to androgen receptor binding sites in the genome, and androgen receptor-driven oncogenic gene fusions. New agents such as enzalutamide, EPI-001, androgen receptor-specific peptidomimetics, novel HSP90 inhibitors, and PARP inhibitors, as well as new approaches to cotargeting the androgen receptor pathway, point to the potential for more complete and durable control of androgen receptor-mediated growth. PMID:24305618

  4. Snake Cytotoxins Bind to Membranes via Interactions with Phosphatidylserine Head Groups of Lipids

    PubMed Central

    Konshina, Anastasia G.; Boldyrev, Ivan A.; Utkin, Yuri N.; Omel'kov, Anton V.; Efremov, Roman G.

    2011-01-01

    The major representatives of Elapidae snake venom, cytotoxins (CTs), share similar three-fingered fold and exert diverse range of biological activities against various cell types. CT-induced cell death starts from the membrane recognition process, whose molecular details remain unclear. It is known, however, that the presence of anionic lipids in cell membranes is one of the important factors determining CT-membrane binding. In this work, we therefore investigated specific interactions between one of the most abundant of such lipids, phosphatidylserine (PS), and CT 4 of Naja kaouthia using a combined, experimental and modeling, approach. It was shown that incorporation of PS into zwitterionic liposomes greatly increased the membrane-damaging activity of CT 4 measured by the release of the liposome-entrapped calcein fluorescent dye. The CT-induced leakage rate depends on the PS concentration with a maximum at approximately 20% PS. Interestingly, the effects observed for PS were much more pronounced than those measured for another anionic lipid, sulfatide. To delineate the potential PS binding sites on CT 4 and estimate their relative affinities, a series of computer simulations was performed for the systems containing the head group of PS and different spatial models of CT 4 in aqueous solution and in an implicit membrane. This was done using an original hybrid computational protocol implementing docking, Monte Carlo and molecular dynamics simulations. As a result, at least three putative PS-binding sites with different affinities to PS molecule were delineated. Being located in different parts of the CT molecule, these anion-binding sites can potentially facilitate and modulate the multi-step process of the toxin insertion into lipid bilayers. This feature together with the diverse binding affinities of the sites to a wide variety of anionic targets on the membrane surface appears to be functionally meaningful and may adjust CT action against different types of

  5. Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide

    PubMed Central

    Gil, Carles; Dorca-Arévalo, Jonatan; Blasi, Juan

    2015-01-01

    Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology. PMID:26452234

  6. Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide.

    PubMed

    Gil, Carles; Dorca-Arévalo, Jonatan; Blasi, Juan

    2015-01-01

    Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology. PMID:26452234

  7. Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

    PubMed Central

    Nanda, Hirsh; Fang, Xianyang; Wen, Yi; Barros, Marilia; Wang, Yun-Xing; Rein, Alan; Vogt, Volker M.

    2015-01-01

    ABSTRACT Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactions in vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particles in vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interaction in vitro, either by directly contacting acidic lipids or by promoting Gag multimerization. IMPORTANCE Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible

  8. Further characterization of ribosome binding to thylakoid membranes. [Pisum sativum

    SciTech Connect

    Hurewitz, J.; Jagendorf, A.T.

    1987-05-01

    Previous work indicated more polysomes bound to pea (Pisum sativum cv Progress No. 9) thylakoids in light than in the dark, in vivo. With isolated intact chloroplasts incubated in darkness, addition of MgATP had no effect but 24 to 74% more RNA was thylakoid-bound at pH 8.3 than at pH 7. Thus, the major effect of light on ribosome-binding in vivo may be due to higher stroma pH. In isolated pea chloroplasts, initiation inhibitors (pactamycin and kanamycin) decreased the extent of RNA binding, and elongation inhibitors (lincomycin and streptomycin) increased it. Thus, cycling of ribosomes is controlled by translation, initiation, and termination. Bound RNA accounted for 19 to 24% of the total chloroplast RNA and the incorporation of (/sup 3/H)leucine into thylakoids was proportional to the amount of this bound RNA. These data support the concept that stroma ribosomes are recruited into thylakoid polysomes, which are active in synthesizing thylakoid proteins.

  9. Identification and purification of a calcium-binding protein in hepatic nuclear membranes.

    PubMed

    Gilchrist, J S; Pierce, G N

    1993-02-25

    Recent evidence suggests that nuclei possess Ca2+ transport mechanisms to regulate nucleoplasmic/cytosolic Ca2+ gradients. We, therefore, investigated the possibility that Ca(2+)-binding proteins may also exist within the nucleus. Electrophoretic analysis revealed the presence of an acidic 93-kDa protein (p93) in the membranes of isolated nuclei. p93 stained blue with "Stains-All" in SDS-polyacrylamide gels and was the major 45Ca(2+)- and ruthenium red-binding nuclear envelope protein in electroblot overlays. p93 was resistant to extraction by 6 M urea but was solubilized in 2% Triton X-100. Citric acid was highly effective in removing the outer nuclear membrane (ER) with concomitant reduction (< 10-fold) of mannose-6-phosphatase activity, but not p93. 45Ca(2+)-binding assays of purified p93 revealed the presence of high capacity Ca(2+)-binding sites comparable to calreticulin. This evidence strongly suggests that p93 is a major Ca(2+)-binding protein of the inner nuclear envelope membrane. Partial amino acid sequence analysis revealed that p93 was close to 100% homologous with a recently identified ER Ca(2+)-binding protein known as calnexin. It is likely, therefore, that p93 is calnexin. However, mild CHAPS detergent treatment of nuclear envelopes and ER revealed distinctly different solubility properties of each membrane for the extraction of p93. This, together with the citrate data, strongly suggests that p93/calnexin, in isolated nuclear envelopes, is mostly bound to the inner membrane. It is possible that p93 may be involved with the regulation of Ca2+ transients between the nucleoplasm and perinuclear space. PMID:8440713

  10. Did Androgen-Binding Protein Paralogs Undergo Neo- and/or Subfunctionalization as the Abp Gene Region Expanded in the Mouse Genome?

    PubMed Central

    Karn, Robert C.; Chung, Amanda G.; Laukaitis, Christina M.

    2014-01-01

    The Androgen-binding protein (Abp) region of the mouse genome contains 30 Abpa genes encoding alpha subunits and 34 Abpbg genes encoding betagamma subunits, their products forming dimers composed of an alpha and a betagamma subunit. We endeavored to determine how many Abp genes are expressed as proteins in tears and saliva, and as transcripts in the exocrine glands producing them. Using standard PCR, we amplified Abp transcripts from cDNA libraries of C57BL/6 mice and found fifteen Abp gene transcripts in the lacrimal gland and five in the submandibular gland. Proteomic analyses identified proteins corresponding to eleven of the lacrimal gland transcripts, all of them different from the three salivary ABPs reported previously. Our qPCR results showed that five of the six transcripts that lacked corresponding proteins are expressed at very low levels compared to those transcripts with proteins. We found 1) no overlap in the repertoires of expressed Abp paralogs in lacrimal gland/tears and salivary glands/saliva; 2) substantial sex-limited expression of lacrimal gland/tear expressed-paralogs in males but no sex-limited expression in females; and 3) that the lacrimal gland/tear expressed-paralogs are found exclusively in ancestral clades 1, 2 and 3 of the five clades described previously while the salivary glands/saliva expressed-paralogs are found only in clade 5. The number of instances of extremely low levels of transcription without corresponding protein production in paralogs specific to tears and saliva suggested the role of subfunctionalization, a derived condition wherein genes that may have been expressed highly in both glands ancestrally were down-regulated subsequent to duplication. Thus, evidence for subfunctionalization can be seen in our data and we argue that the partitioning of paralog expression between lacrimal and salivary glands that we report here occurred as the result of adaptive evolution. PMID:25531410

  11. Binding of isolectins from red kidney bean (Phaseolus vulgaris) to purified rat brush-border membranes.

    PubMed

    Boldt, D H; Banwell, J G

    1985-12-13

    Ingestion of red kidney bean phytohemagglutinin causes impaired growth and intestinal malabsorption, and facilitates bacterial colonization in the small intestine of weanling rats. We have studied interactions of the highly purified phytohemagglutinin erythroagglutinating (E4) and mitogenic (L4) isolectins with microvillous membrane vesicles prepared from rat small intestines. E4 and L4 were radioiodinated with 125I by the chloramine-T technique. E4 and L4 isolectins both bound to microvillous membrane vesicles. Binding was saturable and reversible. Each mg of membrane protein bound 744 +/- 86 micrograms E4 and 213 +/- 21 micrograms L4. The apparent Ka for E4 and L4 binding was 2.5 x 10(-6) and 13.0 x 10(-6) M-1, respectively. Binding of each 125I-labelled isolectin was abolished by 100-fold excess of unlabelled isolectin. In each case binding also was inhibited by appropriate oligosaccharide inhibitors, indicating that isolectin-microvillous membrane interactions were mediated by carbohydrate recognition. Patterns of saccharide inhibition of isolectin binding were different for E4 and L4. Competitive binding experiments demonstrated mutual noncompetitive inhibition of E4 and L4 binding consistent with steric hindrance. Therefore, E4 and L4 each bound to its own set of receptors. Based on the known saccharide specificities of E4 and L4, these data indicate that there are differences in expression of complex asparagine-linked biantennary and tri- or tetraantennary oligosaccharides at the microvillous surface. The data also provide the possibility that direct interactions of one or more phytohemagglutinin isolectins with intestinal mucosa in vivo may contribute to the antinutritional effects associated with ingestion of crude red kidney beans. PMID:4063394

  12. Membrane Cholesterol Modulates the Outward Facing Conformation of the Dopamine Transporter and Alters Cocaine Binding*

    PubMed Central

    Hong, Weimin C.; Amara, Susan G.

    2010-01-01

    Clearance of synaptically released dopamine is regulated by the plasmalemmal dopamine transporter (DAT), an integral membrane protein that resides within a complex lipid milieu. Here we demonstrate that cholesterol, a major component of the lipid bilayer, can modulate the conformation of DAT and alter cocaine binding to DAT. In striatal synaptosomes and transfected cells, DAT was in cholesterol-rich membrane fractions after mild detergent extraction. After increasing the membrane cholesterol content by treatment of water-soluble cholesterol (cholesterol mixed with methyl-β-cyclodextrin), we observed an increase in DAT binding Bmax values for cocaine analogs [3H]WIN35428 and [125I]RTI-55, but similar levels of DAT proteins on the cell surface were shown by surface biotinylation assays. Membrane cholesterol addition also markedly enhanced the accessibility of cysteine sulfhydryl moieties in DAT as probed by a membrane-impermeable maleimide-biotin conjugate. We identified cysteine 306, a juxtamembrane residue on transmembrane domain 6 (TM6) of DAT, as the intrinsic residue exhibiting enhanced reactivity. Similar effects on DAT cysteine accessibility and radioligand binding were observed with addition of zinc, a reagent known to promote the outward facing conformation of DAT. Using substituted cysteine mutants on various positions likely to be extracellular, we identified additional residues located on TM1, TM6, TM7, and TM12 of DAT that are sensitive to alterations in the membrane cholesterol content. Our findings in transfected cells and native tissues support the hypothesis that DAT adopts an outward facing conformation in a cholesterol-rich membrane environment, suggesting a novel modulatory role of the surrounding membrane lipid milieu on DAT function. PMID:20688912

  13. Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes.

    PubMed

    Sezgin, Erdinc; Levental, Ilya; Grzybek, Michal; Schwarzmann, Günter; Mueller, Veronika; Honigmann, Alf; Belov, Vladimir N; Eggeling, Christian; Coskun, Unal; Simons, Kai; Schwille, Petra

    2012-07-01

    Several simplified membrane models featuring coexisting liquid disordered (Ld) and ordered (Lo) lipid phases have been developed to mimic the heterogeneous organization of cellular membranes, and thus, aid our understanding of the nature and functional role of ordered lipid-protein nanodomains, termed "rafts". In spite of their greatly reduced complexity, quantitative characterization of local lipid environments using model membranes is not trivial, and the parallels that can be drawn to cellular membranes are not always evident. Similarly, various fluorescently labeled lipid analogs have been used to study membrane organization and function in vitro, although the biological activity of these probes in relation to their native counterparts often remains uncharacterized. This is particularly true for raft-preferring lipids ("raft lipids", e.g. sphingolipids and sterols), whose domain preference is a strict function of their molecular architecture, and is thus susceptible to disruption by fluorescence labeling. Here, we analyze the phase partitioning of a multitude of fluorescent raft lipid analogs in synthetic Giant Unilamellar Vesicles (GUVs) and cell-derived Giant Plasma Membrane Vesicles (GPMVs). We observe complex partitioning behavior dependent on label size, polarity, charge and position, lipid headgroup, and membrane composition. Several of the raft lipid analogs partitioned into the ordered phase in GPMVs, in contrast to fully synthetic GUVs, in which most raft lipid analogs mis-partitioned to the disordered phase. This behavior correlates with the greatly enhanced order difference between coexisting phases in the synthetic system. In addition, not only partitioning, but also ligand binding of the lipids is perturbed upon labeling: while cholera toxin B binds unlabeled GM1 in the Lo phase, it binds fluorescently labeled GMI exclusively in the Ld phase. Fluorescence correlation spectroscopy (FCS) by stimulated emission depletion (STED) nanoscopy on intact

  14. Membrane cholesterol modulates the outward facing conformation of the dopamine transporter and alters cocaine binding.

    PubMed

    Hong, Weimin C; Amara, Susan G

    2010-10-15

    Clearance of synaptically released dopamine is regulated by the plasmalemmal dopamine transporter (DAT), an integral membrane protein that resides within a complex lipid milieu. Here we demonstrate that cholesterol, a major component of the lipid bilayer, can modulate the conformation of DAT and alter cocaine binding to DAT. In striatal synaptosomes and transfected cells, DAT was in cholesterol-rich membrane fractions after mild detergent extraction. After increasing the membrane cholesterol content by treatment of water-soluble cholesterol (cholesterol mixed with methyl-β-cyclodextrin), we observed an increase in DAT binding B(max) values for cocaine analogs [(3)H]WIN35428 and [(125)I]RTI-55, but similar levels of DAT proteins on the cell surface were shown by surface biotinylation assays. Membrane cholesterol addition also markedly enhanced the accessibility of cysteine sulfhydryl moieties in DAT as probed by a membrane-impermeable maleimide-biotin conjugate. We identified cysteine 306, a juxtamembrane residue on transmembrane domain 6 (TM6) of DAT, as the intrinsic residue exhibiting enhanced reactivity. Similar effects on DAT cysteine accessibility and radioligand binding were observed with addition of zinc, a reagent known to promote the outward facing conformation of DAT. Using substituted cysteine mutants on various positions likely to be extracellular, we identified additional residues located on TM1, TM6, TM7, and TM12 of DAT that are sensitive to alterations in the membrane cholesterol content. Our findings in transfected cells and native tissues support the hypothesis that DAT adopts an outward facing conformation in a cholesterol-rich membrane environment, suggesting a novel modulatory role of the surrounding membrane lipid milieu on DAT function. PMID:20688912

  15. Binding of Hemagglutinin and Influenza Virus to a Peptide-Conjugated Lipid Membrane

    PubMed Central

    Matsubara, Teruhiko; Shibata, Rabi; Sato, Toshinori

    2016-01-01

    Hemagglutinin (HA) plays an important role in the first step of influenza virus (IFV) infection because it initiates the binding of the virus to the sialylgalactose linkages of the receptors on the host cells. We herein demonstrate that a HA-binding peptide immobilized on a solid support is available to bind to HA and IFV. We previously obtained a HA-binding pentapeptide (Ala-Arg-Leu-Pro-Arg), which was identified by phage-display selection against HAs from random peptide libraries. This peptide binds to the receptor-binding site of HA by mimicking sialic acid. A peptide-conjugated lipid (pep-PE) was chemically synthesized from the peptide and a saturated phospholipid. A lipid bilayer composed of pep-PE and an unsaturated phospholipid (DOPC) was immobilized on a mica plate; and the interaction between HA and the pep-PE/DOPC membrane was investigated using atomic force microscopy. The binding of IFV to the pep-PE/DOPC membrane was detected by an enzyme-linked immunosorbent assay and real-time reverse transcription PCR. Our results indicate that peptide-conjugated lipids are a useful molecular device for the detection of HA and IFV. PMID:27092124

  16. The impact of cell-penetrating peptides on membrane bilayer structure during binding and insertion.

    PubMed

    Hirst, Daniel J; Lee, Tzong-Hsien; Kulkarni, Ketav; Wilce, Jacqueline A; Aguilar, Marie-Isabel

    2016-08-01

    We have studied the effect of penetratin and a truncated analogue on the bilayer structure using dual polarisation interferometry, to simultaneously measure changes in mass per unit area and birefringence (an optical parameter representing bilayer order) with high sensitivity during the binding and dissociation from the membrane. Specifically, we studied penetratin (RQIKIWFQNRRMKWKK), along with a shortened and biotinylated version known as R8K-biotin (RRMKWKKK(Biotin)-NH2). Overall both peptides bound only weakly to the neutral DMPC and POPC bilayers, while much higher binding was observed for the anionic DMPC/DMPG and POPC/POPG. The binding of penetratin to gel-phase DMPC/DMPG was adequately represented by a two-state model, whereas on the fluid-phase POPC/POPG it exhibited a distinctly different binding pattern, best represented by a three-state kinetic model. However, R8K-biotin did not bind well to DMPC/DMPG and showed a more transitory and superficial binding to POPC/POPG. Comparing the modelling results for both peptides binding to POPC/POPG suggests an important role for a securely bound intermediate prior to penetratin insertion and translocation. Overall these results further elucidate the mechanism of penetratin, and provide another example of the significance of the ability of DPI to measure structural changes and the use of kinetic analysis to investigate the stages of peptide-membrane interactions. PMID:27163492

  17. Binding of human plasminogen to basement-membrane (type IV) collagen.

    PubMed

    Stack, M S; Moser, T L; Pizzo, S V

    1992-05-15

    Plasminogen, the zymogen form of the serine proteinase plasmin, has been implicated in numerous physiological and pathological processes involving extracellular-matrix remodelling. We have previously demonstrated that the activation of plasminogen catalysed by tissue plasminogen activator is dramatically stimulated in the presence of basement-membrane-specific type IV collagen [Stack, Gonzalez-Gronow & Pizzo (1990) Biochemistry 29, 4966-4970]. The present paper describes the binding of plasminogen to type IV collagen. Plasminogen binds to both the alpha 1(IV) and alpha 2(IV) chains of basement-membrane collagen, with binding to the alpha 2(IV) chain preferentially inhibited by 6-aminohexanoic acid. This binding is specific and saturable, with Kd,app. values of 11.5 and 12.7 nM for collagen and gelatin respectively. Although collagen also binds to immobilized plasminogen, this interaction is unaffected by 6-aminohexanoic acid. Limited elastase proteolysis of plasminogen generated distinct collagen-binding fragments, which were identified as the kringle 1-3 and kringle 4 domains. No binding of collagen to mini-plasminogen was observed. These studies demonstrate a specific interaction between plasminogen and type IV collagen and provide further evidence for regulation of plasminogen activation by protein components of the extracellular matrix. PMID:1599390

  18. Binding of Hemagglutinin and Influenza Virus to a Peptide-Conjugated Lipid Membrane.

    PubMed

    Matsubara, Teruhiko; Shibata, Rabi; Sato, Toshinori

    2016-01-01

    Hemagglutinin (HA) plays an important role in the first step of influenza virus (IFV) infection because it initiates the binding of the virus to the sialylgalactose linkages of the receptors on the host cells. We herein demonstrate that a HA-binding peptide immobilized on a solid support is available to bind to HA and IFV. We previously obtained a HA-binding pentapeptide (Ala-Arg-Leu-Pro-Arg), which was identified by phage-display selection against HAs from random peptide libraries. This peptide binds to the receptor-binding site of HA by mimicking sialic acid. A peptide-conjugated lipid (pep-PE) was chemically synthesized from the peptide and a saturated phospholipid. A lipid bilayer composed of pep-PE and an unsaturated phospholipid (DOPC) was immobilized on a mica plate; and the interaction between HA and the pep-PE/DOPC membrane was investigated using atomic force microscopy. The binding of IFV to the pep-PE/DOPC membrane was detected by an enzyme-linked immunosorbent assay and real-time reverse transcription PCR. Our results indicate that peptide-conjugated lipids are a useful molecular device for the detection of HA and IFV. PMID:27092124

  19. Light-chain binding sites on renal brush-border membranes

    SciTech Connect

    Batuman, V.; Dreisbach, A.W.; Cyran, J.

    1990-05-01

    Immunoglobulin light chains are low-molecular-weight proteins filtered at the renal glomerulus and catabolized within the proximal tubular epithelium. Excessive production and urinary excretion of light chains are associated with renal dysfunction. They also interfere with proximal renal tubule epithelial functions in vitro. We studied the binding of 125I-labeled kappa- and lambda-light chains, obtained from the urine of multiple myeloma patients, to rat and human renal proximal tubular brush-border membranes. Light-chain binding to brush borders was also demonstrated immunologically by flow cytometry. Computer analysis of binding data was consistent with presence of a single class of low-affinity, high-capacity, non-cooperative binding sites with relative selectivity for light chains on both rat and human kidney brush-border membranes. The dissociation constants of light chains ranged from 1.6 X 10(-5) to 1.2 X 10(-4) M, and maximum binding capacity ranged from 4.7 +/- 1.3 X 10(-8) to 8.0 +/- 0.9 X 10(-8) (SD) mol/mg protein at 25 degrees C. Kappa- and lambda-light chains competed with each other for binding with comparable affinity constants. Competition by albumin and beta-lactoglobulin, however, was much weaker, suggesting relative site selectivity for light chains. These binding sites probably function as endocytotic receptors for light chains and possibly other low-molecular-weight proteins.

  20. PLACENTAL EXPRESSION OF THE MEMBRANE FORM OF FOLATE BINDING PROTEIN DURING PREGNANCY IN SWINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous experiments indicated that secreted (s) and membrane (m) forms of folate binding protein (FBP) are present in the intrauterine environment of the pig. Our studies indicated that the two forms were produced sequentially; the secreted form was present in the intrauterine glands until Day 20 o...

  1. Defining the structural characteristics of annexin V binding to a mimetic apoptotic membrane.

    PubMed

    Lu, Jingxiong; Le Brun, Anton P; Chow, Seong Hoong; Shiota, Takuya; Wang, Bo; Lin, Tsung-Wu; Liu, Guei-Sheung; Shen, Hsin-Hui

    2015-12-01

    Annexin V is of crucial importance for detection of the phosphatidylserine of apoptotic cell membranes. However, the manner in which different amounts of phosphatidylserine at the membrane surface at different stages of apoptosis contribute to binding of annexin V is unclear. We have used a quartz crystal microbalance combined with dissipative monitoring (QCM-D) and neutron reflectivity to characterize binding of human annexin V to supported bilayers of different phospholipid composition. We created model apoptotic bilayers of 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine and 1-palmitoyl-2-oleoyl-sn-glycerophosphoserine (POPS) in the ratios 19:1, 9:1, 6.7:1, 4:1, 3:1, and 2:1 (w/w) in the presence of 2.5 mM CaCl2. QCM-D data revealed that annexin V bound less to supported fluid lipid bilayers with higher POPS content (>25 % POPS). Neutron reflectivity was used to further characterize the detailed composition of lipid bilayers with membrane-bound annexin V. Analysis confirmed less annexin V binding with higher POPS content, that bound annexin V formed a discrete layer above the lipid bilayer with little effect on the overall structure of the membrane, and that the thickness and volume fraction of the annexin V layer varied with POPS content. From these results we show that the POPS content of the outer surface of lipid bilayers affects the structure of membrane-bound annexin V. PMID:26271933

  2. Membrane proteins bind lipids selectively to modulate their structure and function

    PubMed Central

    Allison, Timothy M.; Ulmschneider, Martin B.; Degiacomi, Matteo T.; Baldwin, Andrew J.; Robinson, Carol V.

    2014-01-01

    Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments1-7 and that lipids can bind to specific sites, for example in potassium channels8. Fundamental questions remain however regarding the extent of membrane protein selectivity toward lipids. Here we report a mass spectrometry (MS) approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL), aquaporin Z (AqpZ), and the ammonia channel (AmtB) using ion mobility MS (IM-MS), which reports gas-phase collision cross sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas-phase. By resolving lipid-bound states we then rank bound lipids based on their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Results show that lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability, the highest-ranking lipid however is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation9. AqpZ is also stabilized by many lipids with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays, we discover that cardiolipin modulates AqpZ function. Analogous experiments identify AmtB as being highly selective for phosphatidylglycerol prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that reposition AmtB residues to interact with the lipid bilayer. Overall our results demonstrate that resistance to unfolding correlates with specific lipid-binding events enabling distinction of lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these

  3. Structural and Molecular Determinants of Membrane Binding by the HIV-1 Matrix Protein.

    PubMed

    Mercredi, Peter Y; Bucca, Nadine; Loeliger, Burk; Gaines, Christy R; Mehta, Mansi; Bhargava, Pallavi; Tedbury, Philip R; Charlier, Landry; Floquet, Nicolas; Muriaux, Delphine; Favard, Cyril; Sanders, Charles R; Freed, Eric O; Marchant, Jan; Summers, Michael F

    2016-04-24

    Assembly of HIV-1 particles is initiated by the trafficking of viral Gag polyproteins from the cytoplasm to the plasma membrane, where they co-localize and bud to form immature particles. Membrane targeting is mediated by the N-terminally myristoylated matrix (MA) domain of Gag and is dependent on the plasma membrane marker phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Recent studies revealed that PI(4,5)P2 molecules containing truncated acyl chains [tr-PI(4,5)P2] are capable of binding MA in an "extended lipid" conformation and promoting myristoyl exposure. Here we report that tr-PI(4,5)P2 molecules also readily bind to non-membrane proteins, including HIV-1 capsid, which prompted us to re-examine MA-PI(4,5)P2 interactions using native lipids and membrane mimetic liposomes and bicelles. Liposome binding trends observed using a recently developed NMR approach paralleled results of flotation assays, although the affinities measured under the equilibrium conditions of NMR experiments were significantly higher. Native PI(4,5)P2 enhanced MA binding to liposomes designed to mimic non-raft-like regions of the membrane, suggesting the possibility that binding of the protein to disordered domains may precede Gag association with, or nucleation of, rafts. Studies with bicelles revealed a subset of surface and myr-associated MA residues that are sensitive to native PI(4,5)P2, but cleft residues that interact with the 2'-acyl chains of tr-PI(4,5)P2 molecules in aqueous solution were insensitive to native PI(4,5)P2 in bicelles. Our findings call to question extended-lipid MA:membrane binding models, and instead support a model put forward from coarse-grained simulations indicating that binding is mediated predominantly by dynamic, electrostatic interactions between conserved basic residues of MA and multiple PI(4,5)P2 and phosphatidylserine molecules. PMID:26992353

  4. Novel mechanisms of intracellular cholesterol transport: oxysterol-binding proteins and membrane contact sites.

    PubMed

    Du, Ximing; Brown, Andrew J; Yang, Hongyuan

    2015-08-01

    Cholesterol is an essential membrane constituent, and also plays a key role in cell signalling. Within a cell, how cholesterol is transported and how its heterogeneous distribution is maintained are poorly understood. Recent advances have identified novel pathways and regulators of cholesterol trafficking. Sterol transfer by lipid-binding proteins, such as OSBP (oxysterol-binding protein), coupled with phosphatidylinositol 4-phosphate exchange at membrane contact sites (MCSs) has emerged as a new theme of cholesterol transport between organellar membranes. Moreover, a previously unappreciated role of peroxisomes in cholesterol trafficking has been revealed recently. These discoveries highlight the crucial role of MCSs, or junctions, in facilitating lipid movement, and provide mechanistic insights into how cholesterol is sorted in cells. PMID:25932595

  5. Influence of organelle geometry on the apparent binding kinetics of peripheral membrane proteins

    NASA Astrophysics Data System (ADS)

    Hoffmann, Julia; Fickentscher, Rolf; Weiss, Matthias

    2015-02-01

    Information processing in living cells frequently involves an exchange of peripheral membrane proteins between the cytosol and organelle membranes. The typical time scale τ of these association-dissociation cycles is commonly quantified in vivo via fluorescence recovery after photobleaching (FRAP). Contrary to common assumptions, we show here that τ values determined by FRAP depend on the size and number of target structures. Hence, FRAP times alone are insufficient to draw conclusions about the proteins' binding kinetics. In contrast, extracting primary molecular association and dissociation rates from FRAP approaches provides a size-independent and therefore robust measure for the proteins' binding kinetics. We support our theoretical considerations with experiments on the small GTPase Arf-1 that transiently associates with Golgi membranes: While Arf-1 recovery times in untreated cells and in cells with disrupted microtubules are significantly different, the molecular kinetic rates are shown to be the same in both cases.

  6. A functional NMR for membrane proteins: dynamics, ligand binding, and allosteric modulation.

    PubMed

    Oxenoid, Kirill; Chou, James J

    2016-05-01

    By nature of conducting ions, transporting substrates and transducing signals, membrane channels, transporters and receptors are expected to exhibit intrinsic conformational dynamics. It is therefore of great interest and importance to understand the various properties of conformational dynamics acquired by these proteins, for example, the relative population of states, exchange rate, conformations of multiple states, and how small molecule ligands modulate the conformational exchange. Because small molecule binding to membrane proteins can be weak and/or dynamic, structural characterization of these effects is very challenging. This review describes several NMR studies of membrane protein dynamics, ligand-induced conformational rearrangements, and the effect of ligand binding on the equilibrium of conformational exchange. The functional significance of the observed phenomena is discussed. PMID:26928605

  7. GTPase-dependent signaling in bacteria: characterization of a membrane-binding site for era in Escherichia coli.

    PubMed Central

    Lin, Y P; Sharer, J D; March, P E

    1994-01-01

    Era is an Escherichia coli GTPase that is essential for cell viability and is peripherally associated with the cytoplasmic membrane. Both immunoelectron microscopy and subcellular-fractionation experiments have shown that Era is present in cytoplasmic as well as membrane-associated pools. These data led to speculation that the mechanism of action of Era may require cycling between membrane and cytoplasmic sites. In order to investigate this possibility, an in vitro binding assay was developed to characterize the binding of Era to membrane fractions. Competition and saturation binding experiments suggest that a site that is specific for Era and capable of binding up to 5 ng of Era per microgram of membrane protein is present in membrane preparations. The binding curve is complex, indicating that multiple equilibria describe the interaction. The binding of Era to this putative receptor is dependent on guanine nucleotides; binding cannot be measured in the absence of nucleotide, and neither ATP nor UTP can substitute. Subfractionation of cell walls showed that the guanine nucleotide-dependent binding site was present in fractions enriched in cytoplasmic membrane. These data provide evidence that Era may be involved in a GTPase-receptor-coupled membrane-signaling pathway that is essential for growth in E. coli. Images PMID:8282709

  8. Chlamydia trachomatis elementary bodies possess proteins which bind to eucaryotic cell membranes

    SciTech Connect

    Wenman, W.M.; Meuser, R.U.

    1986-02-01

    Chlamydia trachomatis proteins were electrophoresed and then transferred to nitrocellulose paper to detect chlamydial proteins which bind to eucaryotic cell membranes. Resolved polypeptides of C. trachomatis serovars J and L/sub 2/ were reacted with iodinated HeLa cell membranes and autoradiographed. Infectious elementary bodies of both serovars possess 31,000- and 18,000-dalton proteins which bind to HeLa cells. In contrast, noninfectious reticulate bodies do not possess eucaryotic cell-binding proteins. Both proteins are antigenic when reacted with hyperimmune rabbit antisera in immunoblots and antisera raised against the 31,000- and 18,000-dalton proteins are inhibitory to chlamydia-host cell association. In addition, these antisera exhibit neutralizing activity. These data suggest that these putative chlamydial adhesions play a key role in the early steps of chlamydia-host cell interaction and that antibody directed against them may be protective.

  9. Rotenone Activates Phagocyte NADPH Oxidase through Binding to Its Membrane Subunit gp91phox

    PubMed Central

    Zhou, Hui; Zhang, Feng; Chen, Shih-heng; Zhang, Dan; Wilson, Belinda; Hong, Jau-shyong; Gao, Hui-Ming

    2011-01-01

    Rotenone, a widely used pesticide, reproduces Parkinsonism in rodents and associates with increased risk for Parkinson’s disease. We previously reported rotenone increased superoxide production through stimulating microglial phagocyte NADPH oxidase (PHOX). The present study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91phox, the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91phox. Functional studies showed both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91phox/p22phox) and cytosolic subunits (p67phox and p47phox). Rotenone-elicited extracellular superoxide release in p47phox-deficient macrophages suggested rotenone enabled to activate PHOX through a p47phox-independent mechanism. Increased membrane translocation of p67phox, elevated binding of p67phox to rotenone-treated membrane fractions, and co-immunoprecipitation of p67phox and gp91phox in rotenone-treated wild-type and p47phox-deficient macrophages indicated p67phox played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91phox. Rac1, a Rho-like small GTPase, enhanced p67phox-gp91phox interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91phox; such an interaction triggered membrane translocation of p67phox, leading to PHOX activation and superoxide production. PMID:22094225

  10. A comparative study of glycoproteomes in androgen-sensitive and -independent prostate cancer cell lines.

    PubMed

    Drabik, Anna; Ciołczyk-Wierzbicka, Dorota; Dulińska-Litewka, Joanna; Bodzoń-Kułakowska, Anna; Suder, Piotr; Silberring, Jerzy; Laidler, Piotr

    2014-01-01

    Prostate cancer is one of the most common malignancies in men and is predicted to be the second leading cause of cancer-related deaths. After 6-18 months, hormone ablation treatment results in androgen-independent growth of cancer cells, metastasis and progression. The mechanism of androgen-independent growth of prostatic carcinoma cells is still unknown. Identification of factors that facilitate the transition from androgen-dependent to independent states is crucial in designing future diagnostics and medication strategies. To understand the biochemical meaning of hormone dependency deprivation, glycoproteins enriched profiles were compared between DU145 (hormone non-responding) and LNCaP (hormone responding) prostate cancer cells. These results allow for anticipation on the important role of glycosylation in malignant transformation. Both Tn antigen and complex antennary N-oligosaccharides were recognized. Their occurrence might be involved in the development and progression of tumor, and failure of hormone ablation therapy. Among identified proteins in androgen-sensitive cells nucleolin (P19338) was found that is widely described as apoptosis inhibitor, and also transporter of molecules from the membrane to the cytoplasm or nucleus. In addition, 14-3-3 protein family (P27348, P31946, P61981, P63104, P62258, Q04917, and P31947) was investigated across available databases as it forms stable complexes with glycoproteins. Our studies indicate that isoforms: sigma and eta were found in androgen-dependent prostate cancer cells, while other isoforms were present in androgen non-responding cells. 14-3-3 binding partners are involved in cancer pathogenesis. These findings may contribute to a better understanding of prostate cancer tumorigenesis and to a more efficient prognosis and individual therapy in a future. However, it still remains to be revealed how important those changes are for androgen dependency loss in prostate cancer patients carried out on clinically

  11. Properties of estrogen binding components in the plasma membrane of neurohypophysis in hens and changes in its binding before and after oviposition.

    PubMed

    Takahashi, T; Kawashima, M

    2009-10-01

    The present study was performed to elucidate whether the estrogen binding component regarded as a receptor exists in the plasma membrane fraction of neurohypophysis in hens and whether the binding of receptor changes with relation to oviposition. The specific binding for estradiol-17beta (E2) in the neurohypophysis of hens was demonstrated by the use of radioligand binding assays on the plasma membrane fraction of the tissue. The binding to [3H]E2 had a binding specificity to E2 and diethylstilbestrol, reversibility, and saturation. Scatchard analysis revealed that the binding sites are of a single class. The equilibrium dissociation constant obtained by Scatchard analysis and kinetic analysis suggested a high affinity, and the maximum binding capacity obtained by Scatchard analysis suggested a limited capacity. These properties are characteristics of a receptor, which suggests that an estrogen receptor exists in the plasma membrane of hen neurohypophysis. The equilibrium dissociation constant value of estrogen receptor of the neurohypophysis was not significantly different between laying hens and nonlaying hens, but the maximum binding capacity value was statistically smaller (the binding affinity is higher) in laying hens than in nonlaying hens. The specific binding of estrogen receptor showed a decrease at 1 h after an injection of diethylstilbestrol in nonlaying hens. The specific binding also decreased 3 h before oviposition in laying hens and maintained low value until just after oviposition. The present study suggests that estrogen may act directly on the neurohypophysis during 3 h before oviposition in hens. PMID:19762877

  12. A major integral protein of the plant plasma membrane binds flavin.

    PubMed

    Lorenz, Astrid; Kaldenhoff, Ralf; Hertel, Rainer

    2003-05-01

    Abundant flavin binding sites have been found in membranes of plants and fungi. With flavin mononucleotide-agarose affinity columns, riboflavin-binding activity from microsomes of Cucurbita pepoL. hypocotyls was purified and identified as a specific PIP1-homologous protein of the aquaporin family. Sequences such as gi|2149955 in Phaseolus vulgaris, PIP1b of Arabidopsis thaliana, and NtAQP1 of tobacco are closely related. The identification as a riboflavin-binding protein was confirmed by binding tests with an extract of Escherichia coli cells expressing the tobacco NtAQP1 as well as leaves of transgenic tobacco plants that overexpress NtAQP1 or were inhibited in PIP1 expression by antisense constructs. When binding was assayed in the presence of dithionite, the reduced flavin formed a relatively stable association with the protein. Upon dilution under oxidizing conditions, the adduct was resolved, and free flavin reappeared with a half time of about 30 min. Such an association can also be induced photochemically, with oxidized flavin by blue light at 450 nm, in the presence of an electron donor. Several criteria, localization in the plasma membrane, high abundance, affinity to roseoflavin, and photochemistry, argue for a role of the riboflavin-binding protein PIP1 as a photoreceptor. PMID:12768338

  13. ESCRT-III binding protein MITD1 is involved in cytokinesis and has an unanticipated PLD fold that binds membranes

    PubMed Central

    Hadders, Michael A.; Agromayor, Monica; Obita, Takayuki; Perisic, Olga; Caballe, Anna; Kloc, Magdalena; Lamers, Meindert H.; Williams, Roger L.; Martin-Serrano, Juan

    2012-01-01

    The endosomal sorting complexes required for transport (ESCRT) proteins have a critical function in abscission, the final separation of the daughter cells during cytokinesis. Here, we describe the structure and function of a previously uncharacterized ESCRT-III interacting protein, MIT-domain containing protein 1 (MITD1). Crystal structures of MITD1 reveal a dimer, with a microtubule-interacting and trafficking (MIT) domain at the N terminus and a unique, unanticipated phospholipase D-like (PLD) domain at the C terminus that binds membranes. We show that the MIT domain binds to a subset of ESCRT-III subunits and that this interaction mediates MITD1 recruitment to the midbody during cytokinesis. Depletion of MITD1 causes a distinct cytokinetic phenotype consistent with destabilization of the midbody and abscission failure. These results suggest a model whereby MITD1 coordinates the activity of ESCRT-III during abscission with earlier events in the final stages of cell division. PMID:23045692

  14. Peptide displacement of ( sup 3 H)5-hydroxytryptamine binding to bovine cortical membranes

    SciTech Connect

    Takeuchi, Y.; Root-Bernstein, R.S.; Shih, J.C. )

    1990-12-01

    Chemical studies have demonstrated that peptides such as the encephalitogenic (EAE) peptide of myelin basic protein (MBP) and luteinizing hormone-releasing hormone (LHRH) can bind serotonin (5-hydroxytryptamine, 5-HT) in vitro. The present research was undertaken to determine whether such binding interferes with 5-HT binding to its 5-HT1 receptors on bovine cerebral cortical membranes. EAE peptide and LHRH displaced ({sup 3}H)5-HT with IC50s of 4.0 x 10(-4) and 1.8 x 10(-3) M respectively. MBP itself also showed apparent displacing ability with an IC50 of 6.0 x 10(-5) M, though it also caused aggregation of cortical membranes that might have interfered with normal receptor binding. These results support previous suggestions that the tryptophan peptide region of MBP may act as a 5-HT receptor in the neural system. We also tested the effects of muramyl dipeptide (N-acetyl-muramyl-L-Ala-D-isoGln, MD), a bacterial cell-wall breakdown product that acts as a slow-wave sleep promoter, binds to LHRH and EAE peptide, and competes for 5-HT binding sites on macrophages. It showed no significant displacement of 5-HT binding to cortical membranes (IC50 greater than 10(-1) M), but its D-Ala analogue did (IC50 = 1.7 x 10(-3) M). Thus, it seems likely that the 5-HT-related effects of naturally occurring muramyl peptides are physiologically limited by receptor types.

  15. Scaffold-forming and Adhesive Contributions of Synthetic Laminin-binding Proteins to Basement Membrane Assembly.

    PubMed

    McKee, Karen K; Capizzi, Stephanie; Yurchenco, Peter D

    2009-03-27

    Laminins that possess three short arms contribute to basement membrane assembly by anchoring to cell surfaces, polymerizing, and binding to nidogen and collagen IV. Although laminins containing the alpha4 and alpha5 subunits are expressed in alpha2-deficient congenital muscular dystrophy, they may be ineffective substitutes because they bind weakly to cell surfaces and/or because they lack the third arm needed for polymerization. We asked whether linker proteins engineered to bind to deficient laminins that provide such missing activities would promote basement membrane assembly in a Schwann cell model. A chimeric fusion protein (alphaLNNd) that adds a short arm terminus to laminin through the nidogen binding locus was generated and compared with the dystrophy-ameliorating protein miniagrin (mAgrin) that binds to the laminin coiled-coil dystroglycan and sulfatides. alphaLNNd was found to mediate laminin binding to collagen IV, to bind to galactosyl sulfatide, and to selectively convert alpha-short arm deletion-mutant laminins LmDeltaalphaLN and LmDeltaalphaLN-L4b into polymerizing laminins. This protein enabled polymerization-deficient laminin but not an adhesion-deficient laminin lacking LG domains (LmDeltaLG) to assemble an extracellular matrix on Schwann cell surfaces. mAgrin, on the other hand, enabled LmDeltaLG to form an extracellular matrix on cell surfaces without increasing accumulation of non-polymerizing laminins. These gain-of-function studies reveal distinct polymerization and anchorage contributions to basement membrane assembly in which the three different LN domains mediate the former, and the LG domains provide primary anchorage with secondary contributions from the alphaLN domain. These findings may be relevant for an understanding of the pathogenesis and treatment of laminin deficiency states. PMID:19189961

  16. Interaction of P-aminobenzoic acid with normal and sickel erythrocyte membrane: photoaffinity labelling of the binding sites

    SciTech Connect

    Premachandra, B.R.

    1986-03-05

    Electron microscopic studies revealed that P-Amino benzoic acid (PABA) could prevent eichinocytosis of red cells in vitro. Equilibrium binding studies with right side out membrane vesicles (ROV) revealed a similar number of binding sites (1.2-1.4 ..mu..mol/mg) and Kd (1.4-1.6 mM) values for both normal and sickle cell membranes. /sup 14/C-Azide analogue of PABA was synthesized as a photoaffinity label to probe its sites of interaction on the erythrocyte membranes. Competitive binding studies of PABA with its azide indicated that both the compounds share common binding sites on the membrane surface since a 20 fold excess of azide inhibited PABA binding in a linear fashion. The azide was covalently incorporated into the membrane components only upon irradiation (52-35% of the label found in the proteins and the rest in lipids). Electrophoretic analysis of photolabelled ROV revealed that the azide interacts chiefly with Band 3 protein. PABA inhibited both high and low affinity calcium (Ca) binding sites situated on either surface of the membrane in a non-competitive manner; however, Ca binding stimulated by Mg-ATP was not affected. Ca transport into inside out vesicles was inhibited by PABA; but it did not affect the calcium ATP-ase activity. The authors studies suggest that the mechanism of action of PABA is mediated by its interaction with Band 3 protein (anion channel), calcium channel and calcium binding sites of erythrocyte membrane.

  17. Cell cycle-regulated membrane binding of NuMA contributes to efficient anaphase chromosome separation.

    PubMed

    Zheng, Zhen; Wan, Qingwen; Meixiong, Gerry; Du, Quansheng

    2014-03-01

    Accurate and efficient separation of sister chromatids during anaphase is critical for faithful cell division. It has been proposed that cortical dynein-generated pulling forces on astral microtubules contribute to anaphase spindle elongation and chromosome separation. In mammalian cells, however, definitive evidence for the involvement of cortical dynein in chromosome separation is missing. It is believed that dynein is recruited and anchored at the cell cortex during mitosis by the α subunit of heterotrimeric G protein (Gα)/mammalian homologue of Drosophila Partner of Inscuteable/nuclear mitotic apparatus (NuMA) ternary complex. Here we uncover a Gα/LGN-independent lipid- and membrane-binding domain at the C-terminus of NuMA. We show that the membrane binding of NuMA is cell cycle regulated-it is inhibited during prophase and metaphase by cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation and only occurs after anaphase onset when CDK1 activity is down-regulated. Further studies indicate that cell cycle-regulated membrane association of NuMA underlies anaphase-specific enhancement of cortical NuMA and dynein. By replacing endogenous NuMA with membrane-binding-deficient NuMA, we can specifically reduce the cortical accumulation of NuMA and dynein during anaphase and demonstrate that cortical NuMA and dynein contribute to efficient chromosome separation in mammalian cells. PMID:24371089

  18. Clueless is a conserved ribonucleoprotein that binds the ribosome at the mitochondrial outer membrane

    PubMed Central

    Sen, Aditya; Cox, Rachel T.

    2016-01-01

    ABSTRACT Mitochondrial function is tied to the nucleus, in that hundreds of proteins encoded by nuclear genes must be imported into mitochondria. While post-translational import is fairly well understood, emerging evidence supports that mitochondrial site-specific import, or co-translational import, also occurs. However, the mechanism and the extent to which it is used are not fully understood. We have previously shown Clueless (Clu), a conserved multi-domain protein, associates with mitochondrial outer membrane proteins, including Translocase of outer membrane 20, and genetically and physically interacts with the PINK1–Parkin pathway. The human ortholog of Clu, Cluh, was shown to bind nuclear-encoded mitochondrially destined mRNAs. Here we identify the conserved tetratricopeptide domain of Clu as predominantly responsible for binding mRNA. In addition, we show Clu interacts with the ribosome at the mitochondrial outer membrane. Taken together, these data support a model whereby Clu binds to and mitochondrially targets mRNAs to facilitate mRNA localization to the outer mitochondrial membrane, potentially for site-specific or co-translational import. This role may link the presence of efficient mitochondrial protein import to mitochondrial quality control through the PINK1–Parkin pathway. PMID:26834020

  19. Membrane Environment Can Enhance the Interaction of Glycan Binding Protein to Cell Surface Glycan Receptors

    PubMed Central

    2015-01-01

    The binding of lectins to glycan receptors on the host cell surface is a key step contributing to the virulence and species specificity of most viruses. This is exemplified by the viral protein hemagglutinin (HA) of the influenza A virus, whose binding specificity is modulated by the linkage pattern of terminal sialic acids on glycan receptors of host epithelial cells. Such specificity dictates whether transmission is confined to a particular animal species or jumps between species. Here, we show, using H5N1 avian influenza as a model, that the specific binding of recombinant HA to α2-3 linked sialic acids can be enhanced dramatically by interaction with the surface of the lipid membrane. This effect can be quantitatively accounted for by a two-stage process in which weak association of HA with the membrane surface precedes more specific and tighter binding to the glycan receptor. The weak protein–membrane interaction discovered here in the model system may play an important secondary role in the infection and pathogenesis of the influenza A virus. PMID:24949798

  20. Identification by cross-linking of a beta-bungarotoxin binding polypeptide in chick brain membranes.

    PubMed Central

    Rehm, H; Betz, H

    1983-01-01

    beta-Bungarotoxin (beta-BTX) is a snake venom neurotoxin which inhibits neurotransmitter release from different types of nerve terminals. To identify presynaptic membrane components potentially important in neurosecretion, 125I-labeled beta-BTX (mol. wt. 21 000) was cross-linked to a high-affinity binding site in synaptic membrane fractions of chick brain using the photoactivable cross-linker N-succinimidyl-6(4'-azido-2'-nitrophenylamino)-hexanoate. Electrophoretic analysis of the cross-linked membrane proteins under both reducing and non-reducing conditions revealed a single [125I]beta-BTX-polypeptide adduct of apparent mol. wt. 116 000 (+/- 2000). The labeling of this band was prevented under conditions previously shown to inhibit the binding of [125I]beta-BTX to its high-affinity binding site. It is concluded that the cross-linking procedure identified a polypeptide of the presynaptic binding site for beta-BTX, and that this polypeptide has a mol. wt. of 95 000. Images Fig. 1. Fig. 2. Fig. 3. PMID:6628364

  1. Mode of Ezrin-Membrane Interaction as a Function of PIP2 Binding and Pseudophosphorylation.

    PubMed

    Shabardina, Victoria; Kramer, Corinna; Gerdes, Benjamin; Braunger, Julia; Cordes, Andrea; Schäfer, Jonas; Mey, Ingo; Grill, David; Gerke, Volker; Steinem, Claudia

    2016-06-21

    Ezrin, a protein of the ezrin, radixin, moesin (ERM) family, provides a regulated linkage between the plasma membrane and the cytoskeleton. The hallmark of this linkage is the activation of ezrin by phosphatidylinositol-4,5-bisphosphate (PIP2) binding and a threonine phosphorylation at position 567. To analyze the influence of these activating factors on the organization of ezrin on lipid membranes and the proposed concomitant oligomer-monomer transition, we made use of supported lipid bilayers in conjunction with atomic force microscopy and fluorescence microscopy. Bilayers doped with either PIP2 as the natural receptor lipid of ezrin or a Ni-nitrilotriacetic acid-equipped lipid to bind the proteins via their His6-tags to the lipid membrane were used to bind two different ezrin variants: ezrin wild-type and ezrin T567D mimicking the phosphorylated state. Using a combination of reflectometric interference spectroscopy, atomic force microscopy, and Förster resonance energy transfer experiments, we show that only the ezrin T567D mutant, upon binding to PIP2-containing bilayers, undergoes a remarkable conformational change, which we attribute to an opening of the conformation resulting in monomeric protein on the lipid bilayer. PMID:27332129

  2. Different modes of lipid binding to membrane proteins probed by mass spectrometry.

    PubMed

    Bechara, Chérine; Robinson, Carol V

    2015-04-29

    The realization that the lipid environment is crucial for maintaining the structure and function of membrane proteins prompts new methods to understand lipid interactions. One such method, mass spectrometry, is emerging with the potential to monitor different modes of lipid binding to membrane protein complexes. Initial studies monitored the addition of lipids and deduced the kinetic and thermodynamic effects of lipid binding to proteins. Recent efforts however have focused on identifying lipids already present, explicitly in plugs, annular rings, or cavities. Lipids that bind within these orifices to membrane proteins will have higher residence times than those in the bulk lipid bilayer and consequently can be quantified and characterized by mass spectrometry. In special cases, lipids identified within cavities have been proposed as substrates following activity assays. Alternatively, a gas-phase unfolding protocol can be used to distinguish lipids that are important for stability. These lipids can subsequently be added during crystallization for the characterization of lipid-bound protein complexes. Overall therefore this Perspective provides an overview of recent advances in mass spectrometry, with a particular focus on the distinction of the various modes of lipid binding, and their implications for structure and function as well as new directions that lie ahead. PMID:25860341

  3. Membrane topology and DNA-binding ability of the Streptococcal CpsA protein.

    PubMed

    Hanson, Brett R; Lowe, Beth A; Neely, Melody N

    2011-01-01

    Many streptococcal pathogens require a polysaccharide capsule for survival in the host during systemic infection. The highly conserved CpsA protein is proposed to be a transcriptional regulator of capsule production in streptococci, although the regulatory mechanism is unknown. Hydropathy plots of CpsA predict an integral membrane protein with 3 transmembrane domains and only 27 cytoplasmic residues, whereas other members of the LytR_cpsA_psr protein family are predicted to have a single transmembrane domain. This unique topology, with the short cytoplasmic domain, membrane localization, and large extracellular domain, suggests a novel mechanism of transcriptional regulation. Therefore, to determine the actual membrane topology of CpsA, specific protein domains were fused to beta-galactosidase or alkaline phosphatase. Enzymatic assays confirmed that the predicted membrane topology for CpsA is correct. To investigate how this integral membrane protein may be functioning in regulation of capsule transcription, purified full-length and truncated forms of CpsA were used in electrophoretic mobility shift assays to characterize the ability to bind the capsule operon promoter. Assays revealed that full-length, purified CpsA protein binds specifically to DNA containing the capsule promoter region. Furthermore, the large extracellular domain is not required for DNA binding, but all cytoplasmic regions of CpsA are necessary and sufficient for specific binding to the capsule operon promoter. This is the first demonstration of a member of this protein family interacting with its target DNA. Taken together, CpsA, as well as other members of the LytR_cpsA_psr protein family, appears to utilize a unique mechanism of transcriptional regulation. PMID:21097630

  4. Binding of monovalent alkali metal ions with negatively charged phospholipid membranes.

    PubMed

    Maity, Pabitra; Saha, Baishakhi; Kumar, Gopinatha Suresh; Karmakar, Sanat

    2016-04-01

    We have systematically investigated the effect of various alkali metal ions with negatively charged phospholipid membranes. Size distributions of large unilamellar vesicles have been confirmed using dynamic light scattering. Zeta potential and effective charges per vesicle in the presence of various alkali metal ions have been estimated from the measured electrophoretic mobility. We have determined the intrinsic binding constant from the zeta potential using electrostatic double layer theory. The reasonable and consistent value of the intrinsic binding constant of Na(+), found at moderate NaCl concentration (10-100 mM), indicates that the Gouy-Chapman theory cannot be applied for very high (> 100mM) and very low (< 10 mM) electrolyte concentrations. The isothermal titration calorimetry study has revealed that the net binding heat of interaction of the negatively charged vesicles with monovalent alkali metal ions is small and comparable to those obtained from neutral phosphatidylcholine vesicles. The overall endothermic response of binding heat suggests that interaction is primarily entropy driven. The entropy gain might arise due to the release of water molecules from the hydration layer vicinity of the membranes. Therefore, the partition model which does not include the electrostatic contribution suffices to describe the interaction. The binding constant of Na(+) (2.4 ± 0.1 M(-1)), obtained from the ITC, is in agreement with that estimated from the zeta potential (-2.0 M(-1)) at moderate salt concentrations. Our results suggest that hydration dynamics may play a vital role in the membrane solution interface which strongly affects the ion-membrane interaction. PMID:26802251

  5. Prediction of binding free energy for adsorption of antimicrobial peptide lactoferricin B on a POPC membrane

    NASA Astrophysics Data System (ADS)

    Vivcharuk, Victor; Tomberli, Bruno; Tolokh, Igor S.; Gray, C. G.

    2008-03-01

    Molecular dynamics (MD) simulations are used to study the interaction of a zwitterionic palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayer with the cationic antimicrobial peptide bovine lactoferricin (LFCinB) in a 100 mM NaCl solution at 310 K. The interaction of LFCinB with POPC is used as a model system for studying the details of membrane-peptide interactions, with the peptide selected because of its antimicrobial nature. Seventy-two 3 ns MD simulations, with six orientations of LFCinB at 12 different distances from a POPC membrane, are carried out to determine the potential of mean force (PMF) or free energy profile for the peptide as a function of the distance between LFCinB and the membrane surface. To calculate the PMF for this relatively large system a new variant of constrained MD and thermodynamic integration is developed. A simplified method for relating the PMF to the LFCinB-membrane binding free energy is described and used to predict a free energy of adsorption (or binding) of -1.05±0.39kcal/mol , and corresponding maximum binding force of about 20 pN, for LFCinB-POPC. The contributions of the ions-LFCinB and the water-LFCinB interactions to the PMF are discussed. The method developed will be a useful starting point for future work simulating peptides interacting with charged membranes and interactions involved in the penetration of membranes, features necessary to understand in order to rationally design peptides as potential alternatives to traditional antibiotics.

  6. Accurate quantification of diffusion and binding kinetics of non-integral membrane proteins by FRAP.

    PubMed

    Berkovich, Ronen; Wolfenson, Haguy; Eisenberg, Sharon; Ehrlich, Marcelo; Weiss, Matthias; Klafter, Joseph; Henis, Yoav I; Urbakh, Michael

    2011-11-01

    Non-integral membrane proteins frequently act as transduction hubs in vital signaling pathways initiated at the plasma membrane (PM). Their biological activity depends on dynamic interactions with the PM, which are governed by their lateral and cytoplasmic diffusion and membrane binding/unbinding kinetics. Accurate quantification of the multiple kinetic parameters characterizing their membrane interaction dynamics has been challenging. Despite a fair number of approximate fitting functions for analyzing fluorescence recovery after photobleaching (FRAP) data, no approach was able to cope with the full diffusion-exchange problem. Here, we present an exact solution and matlab fitting programs for FRAP with a stationary Gaussian laser beam, allowing simultaneous determination of the membrane (un)binding rates and the diffusion coefficients. To reduce the number of fitting parameters, the cytoplasmic diffusion coefficient is determined separately. Notably, our equations include the dependence of the exchange kinetics on the distribution of the measured protein between the PM and the cytoplasm, enabling the derivation of both k(on) and k(off) without prior assumptions. After validating the fitting function by computer simulations, we confirm the applicability of our approach to live-cell data by monitoring the dynamics of GFP-N-Ras mutants under conditions with different contributions of lateral diffusion and exchange to the FRAP kinetics. PMID:21810156

  7. Weak Glycolipid Binding of a Microdomain-Tracer Peptide Correlates with Aggregation and Slow Diffusion on Cell Membranes

    PubMed Central

    Lauterbach, Tim; Manna, Manoj; Ruhnow, Maria; Wisantoso, Yudi; Wang, Yaofeng; Matysik, Artur; Oglęcka, Kamila; Mu, Yuguang; Geifman-Shochat, Susana; Wohland, Thorsten; Kraut, Rachel

    2012-01-01

    Organized assembly or aggregation of sphingolipid-binding ligands, such as certain toxins and pathogens, has been suggested to increase binding affinity of the ligand to the cell membrane and cause membrane reorganization or distortion. Here we show that the diffusion behavior of the fluorescently tagged sphingolipid-interacting peptide probe SBD (Sphingolipid Binding Domain) is altered by modifications in the construction of the peptide sequence that both result in a reduction in binding to ganglioside-containing supported lipid membranes, and at the same time increase aggregation on the cell plasma membrane, but that do not change relative amounts of secondary structural features. We tested the effects of modifying the overall charge and construction of the SBD probe on its binding and diffusion behavior, by Surface Plasmon Resonance (SPR; Biacore) analysis on lipid surfaces, and by Fluorescence Correlation Spectroscopy (FCS) on live cells, respectively. SBD binds preferentially to membranes containing the highly sialylated gangliosides GT1b and GD1a. However, simple charge interactions of the peptide with the negative ganglioside do not appear to be a critical determinant of binding. Rather, an aggregation-suppressing amino acid composition and linker between the fluorophore and the peptide are required for optimum binding of the SBD to ganglioside-containing supported lipid bilayer surfaces, as well as for interaction with the membrane. Interestingly, the strength of interactions with ganglioside-containing artificial membranes is mirrored in the diffusion behavior by FCS on cell membranes, with stronger binders displaying similar characteristic diffusion profiles. Our findings indicate that for aggregation-prone peptides, aggregation occurs upon contact with the cell membrane, and rather than giving a stronger interaction with the membrane, aggregation is accompanied by weaker binding and complex diffusion profiles indicative of heterogeneous diffusion

  8. Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity.

    PubMed

    Murashko, Oleg N; Kaberdin, Vladimir R; Lin-Chao, Sue

    2012-05-01

    RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane-protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E-membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1-499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (K(d)) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the "large" domain (amino acids 1-400, consisting of the RNase H, S1, 5'-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 °C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E-membrane interaction enhances the rate of RNA processing and decay. PMID:22509045

  9. Phosphatidylserine-binding protein lactadherin inhibits protein translocation across the ER membrane.

    PubMed

    Yamamoto, Hitoshi; Kida, Yuichiro; Sakaguchi, Masao

    2013-05-10

    Secretory and membrane proteins are translocated across and inserted into the endoplasmic reticulum membrane via translocon channels. To investigate the effect of the negatively-charged phospholipid phosphatidylserine on the translocation of nascent polypeptide chains through the translocon, we used the phosphatidylserine-binding protein lactadherin C2-domain. Lactadherin inhibited targeting of nascent chain to the translocon by signal sequence and the initiation of translocation. Moreover, lactadherin inhibited the movement of the translocating polypeptide chain regardless of the presence or absence of positively-charged residues. Phosphatidylserine might be critically involved in translocon function, but it is not a major determinant for translocation arrest of positively-charged residues. PMID:23583395

  10. Aberrant splicing of androgenic receptor mRNA results in synthesis of a nonfunctional receptor protein in a patient with androgen insensitivity

    SciTech Connect

    Ris-Stalpers, C.; Kuiper, G.G.J.M.; Faber, P.W.; van Rooij, H.C.J.; Degenhart, H.J.; Trapman, J.; Brinkmann, A.O. ); Schweikert, H.U. ); Zegers, N.D. ); Hodgins, M.B. )

    1990-10-01

    Androgen insensitivity is a disorder in which the correct androgen response in an androgen target cell is impaired. The clinical symtpoms of this X chromosome-linked syndrome are presumed to be caused by mutations in the androgen receptor gene. The authors report a G {r arrow} T mutation in the splice donor site of intron 4 of the androgen receptor gene of a 46, XY subject lacking detectable androgen binding to the receptor and with the complete form of androgen insensitivity. This point mutation completely abolishes normal RNA splicing at the exon 4/intron 4 boundary and results in the activation of a cryptic splice donor site in exon 4, which leads to the deletion of 123 nucleotides from the mRNA. Translation of the mutant mRNA results in an androgen receptor protein {approx}5 kDa smaller than the wild type. This mutated androgen receptor protein was unable to bind androgens and unable to activate transcription of an androgen-regulated reporter gene construct. This mutation in the human androgen receptor gene demonstrates the importance of an intact steroid-binding domain for proper androgen receptor functioning in vivo.

  11. A Role for Weak Electrostatic Interactions in Peripheral Membrane Protein Binding.

    PubMed

    Khan, Hanif M; He, Tao; Fuglebakk, Edvin; Grauffel, Cédric; Yang, Boqian; Roberts, Mary F; Gershenson, Anne; Reuter, Nathalie

    2016-03-29

    Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) is a secreted virulence factor that binds specifically to phosphatidylcholine (PC) bilayers containing negatively charged phospholipids. BtPI-PLC carries a negative net charge and its interfacial binding site has no obvious cluster of basic residues. Continuum electrostatic calculations show that, as expected, nonspecific electrostatic interactions between BtPI-PLC and membranes vary as a function of the fraction of anionic lipids present in the bilayers. Yet they are strikingly weak, with a calculated ΔGel below 1 kcal/mol, largely due to a single lysine (K44). When K44 is mutated to alanine, the equilibrium dissociation constant for small unilamellar vesicles increases more than 50 times (∼2.4 kcal/mol), suggesting that interactions between K44 and lipids are not merely electrostatic. Comparisons of molecular-dynamics simulations performed using different lipid compositions reveal that the bilayer composition does not affect either hydrogen bonds or hydrophobic contacts between the protein interfacial binding site and bilayers. However, the occupancies of cation-π interactions between PC choline headgroups and protein tyrosines vary as a function of PC content. The overall contribution of basic residues to binding affinity is also context dependent and cannot be approximated by a rule-of-thumb value because these residues can contribute to both nonspecific electrostatic and short-range protein-lipid interactions. Additionally, statistics on the distribution of basic amino acids in a data set of membrane-binding domains reveal that weak electrostatics, as observed for BtPI-PLC, might be a less unusual mechanism for peripheral membrane binding than is generally thought. PMID:27028646

  12. Hydrophobic ion interactions with membranes. Thermodynamic analysis of tetraphenylphosphonium binding to vesicles.

    PubMed Central

    Flewelling, R F; Hubbell, W L

    1986-01-01

    The thermodynamic properties for the interaction of the hydrophobic ion tetraphenylphosphonium (TPP+) with egg phosphatidylcholine vesicles were studied in detail by equilibrium dialysis and spin label techniques. A partition coefficient of beta = 4.2 + 0.4 x 10(-6) cm (K congruent to 100) was determined. Electrostatic saturation sets in at approximately 600 microM (about one absorbed TPP+ molecule per 100 lipids), and is not screened by salt. The temperature dependence of binding was determined, which reveals that the binding is entropy-driven with a positive (repulsive) enthalpy of binding, a result to be compared with hydrophobic anions in which the binding enthalpy is negative. The membrane dipole potential may be responsible for this binding difference. Activity coefficients are determined and shown to be significantly different from those of most common salts, an important result that should be considered in all hydrophobic ion studies. Comparison of the TPP+ results with those of its anionic structural analogue, tetraphenylboron (TPB-), permits a general analysis of hydrophobic ion interactions with membranes. A theoretical model consistent with the entire set of data is developed in an accompanying article. PMID:3006814

  13. Vitellogenin Recognizes Cell Damage through Membrane Binding and Shields Living Cells from Reactive Oxygen Species*

    PubMed Central

    Havukainen, Heli; Münch, Daniel; Baumann, Anne; Zhong, Shi; Halskau, Øyvind; Krogsgaard, Michelle; Amdam, Gro V.

    2013-01-01

    Large lipid transfer proteins are involved in lipid transportation and diverse other molecular processes. These serum proteins include vitellogenins, which are egg yolk precursors and pathogen pattern recognition receptors, and apolipoprotein B, which is an anti-inflammatory cholesterol carrier. In the honey bee, vitellogenin acts as an antioxidant, and elevated vitellogenin titer is linked to prolonged life span in this animal. Here, we show that vitellogenin has cell and membrane binding activity and that it binds preferentially to dead and damaged cells. Vitellogenin binds directly to phosphatidylcholine liposomes and with higher affinity to liposomes containing phosphatidylserine, a lipid of the inner leaflet of cell membranes that is exposed in damaged cells. Vitellogenin binding to live cells, furthermore, improves cell oxidative stress tolerance. This study can shed more light on why large lipid transfer proteins have a well conserved α-helical domain, because we locate the lipid bilayer-binding ability of vitellogenin largely to this region. We suggest that recognition of cell damage and oxidation shield properties are two mechanisms that allow vitellogenin to extend honey bee life span. PMID:23897804

  14. An immunodominant membrane protein (Imp) of 'Candidatus Phytoplasma mali' binds to plant actin.

    PubMed

    Boonrod, K; Munteanu, B; Jarausch, B; Jarausch, W; Krczal, G

    2012-07-01

    The phytopathogenic, cell-wall-less phytoplasmas exhibit a dual life cycle: they multiply in the phloem of their host plant and in the body of their insect vector. Their membrane proteins are in direct contact with both hosts and are supposed to play a crucial role in the phytoplasma spread within the plant as well as by the insect vector. Three types of nonhomologous but highly abundant and immunodominant membrane proteins (IDP) have been identified within the phytoplasmas: Amp, IdpA, and Imp. Although recent results indicate that Amp is involved in vector specificity interacting with insect proteins such as actin, little is known about the interaction of IDP with the plant. We could demonstrate that transiently expressed Imp of 'Candidatus Phytoplasma mali' as well as the Imp without transmembrane domain (Imp▴Tm) bind with plant actins in vivo. Moreover, in vitro co-sediment and binding assays showed that Escherichia coli-expressed recombinant Imp▴Tm-His binds to both G- and F-actins isolated from rabbit muscle. Transgenic plants expressing Imp- or Imp▴Tm-green fluorescent protein did not exhibit any remarkable change of phenotype compared with the wild-type plant. These results indicate that Imp specifically binds to plant actin and a role of Imp-actin binding in phytoplasma motility is hypothesized. PMID:22432876

  15. Bacillus thuringiensis delta-endotoxin binding to brush border membrane vesicles of rice stem borers.

    PubMed

    Alcantara, Edwin P; Aguda, Remedios M; Curtiss, April; Dean, Donald H; Cohen, Michael B

    2004-04-01

    The receptor binding step in the molecular mode of action of five delta-endotoxins (Cry1Ab, Cry1Ac, Cry1C, Cry2A, and Cry9C) from Bacillus thuringiensis was examined to find toxins with different receptor sites in the midgut of the striped stem borer (SSB) Chilo suppressalis (Walker) and yellow stem borer (YSB) Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae). Homologous competition assays were used to estimate binding affinities (K(com)) of (125)I-labelled toxins to brush border membrane vesicles (BBMV). The SSB BBMV affinities in decreasing order was: Cry1Ab = Cry1Ac > Cry9C > Cry2A > Cry1C. In YSB, the order of decreasing affinities was: Cry1Ac > Cry1Ab > Cry9C = Cry2A > Cry1C. The number of binding sites (B(max)) estimated by homologous competition binding among the Cry toxins did not affect toxin binding affinity (K(com)) to both insect midgut BBMVs. Results of the heterologous competition binding assays suggest that Cry1Ab and Cry1Ac compete for the same binding sites in SSB and YSB. Other toxins bind with weak (Cry1C, Cry2A) or no affinity (Cry9C) to Cry1Ab and Cry1Ac binding sites in both species. Cry2A had the lowest toxicity to 10-day-old SSB and Cry1Ab and Cry1Ac were the most toxic. Taken together, the results of this study show that Cry1Ab or Cry1Ac could be combined with either Cry1C, Cry2A, or Cry9C for more durable resistance in transgenic rice. Cry1Ab should not be used together with Cry1Ac because a mutation in one receptor site could diminish binding of both toxins. PMID:15027071

  16. Isothermal titration calorimetry with micelles: Thermodynamics of inhibitor binding to carnitine palmitoyltransferase 2 membrane protein.

    PubMed

    Perspicace, Samantha; Rufer, Arne C; Thoma, Ralf; Mueller, Francis; Hennig, Michael; Ceccarelli, Simona; Schulz-Gasch, Tanja; Seelig, Joachim

    2013-01-01

    Carnitine palmitoyl transferase 2 (CPT-2) is a key enzyme in the mitochondrial fatty acid metabolism. The active site is comprised of a Y-shaped tunnel with distinct binding sites for the substrate acylcarnitine and the cofactor CoA. We investigated the thermodynamics of binding of four inhibitors directed against either the CoA or the acylcarnitine binding sites using isothermal titration calorimetry (ITC). CPT-2 is a monotopic membrane protein and was solubilized by β-octylglucoside (β-OG) above its critical micellar concentration (CMC) to perform inhibitor titrations in solutions containing detergent micelles. The CMC of β-OG in the presence of inhibitors was measured with ITC and small variations were observed. The inhibitors bound to rat CPT-2 (rCPT-2) with 1:1 stoichiometry and the dissociation constants were in the range of K D = 2-20 μM. New X-ray structures and docking models of rCPT-2 in complex with inhibitors enable an analysis of the thermodynamic data in the context of the interaction observed for the individual binding sites of the ligands. For all ligands the binding enthalpy was exothermic, and enthalpy as well as entropy contributed to the binding reaction, with the exception of ST1326 for which binding was solely enthalpy-driven. The substrate analog ST1326 binds to the acylcarnitine binding site and a heat capacity change close to zero suggests a balance of electrostatic and hydrophobic interactions. An excellent correlation of the thermodynamic (ITC) and structural (X-ray crystallography, models) data was observed suggesting that ITC measurements provide valuable information for optimizing inhibitor binding in drug discovery. PMID:23772395

  17. Isothermal titration calorimetry with micelles: Thermodynamics of inhibitor binding to carnitine palmitoyltransferase 2 membrane protein☆

    PubMed Central

    Perspicace, Samantha; Rufer, Arne C.; Thoma, Ralf; Mueller, Francis; Hennig, Michael; Ceccarelli, Simona; Schulz-Gasch, Tanja; Seelig, Joachim

    2013-01-01

    Carnitine palmitoyl transferase 2 (CPT-2) is a key enzyme in the mitochondrial fatty acid metabolism. The active site is comprised of a Y-shaped tunnel with distinct binding sites for the substrate acylcarnitine and the cofactor CoA. We investigated the thermodynamics of binding of four inhibitors directed against either the CoA or the acylcarnitine binding sites using isothermal titration calorimetry (ITC). CPT-2 is a monotopic membrane protein and was solubilized by β-octylglucoside (β-OG) above its critical micellar concentration (CMC) to perform inhibitor titrations in solutions containing detergent micelles. The CMC of β-OG in the presence of inhibitors was measured with ITC and small variations were observed. The inhibitors bound to rat CPT-2 (rCPT-2) with 1:1 stoichiometry and the dissociation constants were in the range of KD = 2–20 μM. New X-ray structures and docking models of rCPT-2 in complex with inhibitors enable an analysis of the thermodynamic data in the context of the interaction observed for the individual binding sites of the ligands. For all ligands the binding enthalpy was exothermic, and enthalpy as well as entropy contributed to the binding reaction, with the exception of ST1326 for which binding was solely enthalpy-driven. The substrate analog ST1326 binds to the acylcarnitine binding site and a heat capacity change close to zero suggests a balance of electrostatic and hydrophobic interactions. An excellent correlation of the thermodynamic (ITC) and structural (X-ray crystallography, models) data was observed suggesting that ITC measurements provide valuable information for optimizing inhibitor binding in drug discovery. PMID:23772395

  18. Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity

    PubMed Central

    Murashko, Oleg N.; Kaberdin, Vladimir R.; Lin-Chao, Sue

    2012-01-01

    RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane–protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E–membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1–499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (Kd) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the “large” domain (amino acids 1–400, consisting of the RNase H, S1, 5′-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 °C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E–membrane interaction enhances the rate of RNA processing and decay. PMID:22509045

  19. Calculations of distance distributions and probabilities of binding by ligands between parallel plane membranes comprising receptors

    NASA Astrophysics Data System (ADS)

    Plante, Ianik; Devroye, Luc; Cucinotta, Francis A.

    2014-03-01

    Cell communication through biochemical signaling pathways is a key determinant of tissue responses to radiation. Several molecules, such as the transforming growth factor β (TGFβ), are implicated in radiation-induced signaling between cells. Brownian Dynamics (BD) algorithms have recently been used to simulate the interaction of ligands with receptors and to elucidate signal transduction and autocrine loops in ligand-receptors systems. In this paper, we discuss the simulation of particle diffusion and binding kinetics in a space bounded by two parallel plane membranes, using an exact algorithm to sample the propagator (Green’s function) of a particle located between 2 membranes. We also show that the simulation results are independent of the number of time steps used, in accordance with time discretization equations. These simulations could be used to simulate the motion and binding of ligand molecules in a cell culture, and possibly in neuronal synapses.

  20. Isoprenoid modification permits 2',3'-cyclic nucleotide 3'-phosphodiesterase to bind to membranes.

    PubMed

    Braun, P E; De Angelis, D; Shtybel, W W; Bernier, L

    1991-11-01

    The myelination-related enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a relatively abundant protein in the CNS possesses the C-terminal isoprenylation consensus domain found in a small family that includes the ras oncoproteins and their relatives, some G-proteins, and nuclear lamins. We found that CNP, like these other proteins, is modified posttranslationally by an isoprenoid derived from mevalonic acid. It appears that only the smaller of the two CNP isoforms (CNP1) is isoprenylated, but similar modification of CNP2 cannot be excluded. Inhibition of isoprenoid synthesis by Lovastatin blocks the binding of newly synthesized CNP to cell membranes; binding is restored upon addition of mevalonate to the culture medium. This shows that isoprenylation is permissive for the well-known avid association of CNP with membranes. PMID:1666129

  1. Role of fluidity of membranes on the guanyl nucleotide-dependent binding of cholecystokinin-8S to rat brain cortical membranes.

    PubMed

    Rinken, A; Harro, J; Engström, L; Oreland, L

    1998-02-15

    The binding of [3H]cholecystokinin octapeptide (sulphated) ([3H]CCK-8S), an agonist of the cholecystokinin receptors, to rat cortical membranes was fast, specific and saturable, with pH optimum at 6.5-7.0. The divalent cations Mg2+ and Ca2+ clearly enhanced [3H]CCK-8S binding, whereas the monovalent cations Na+ and K+ were inhibitors. Inactivation of the ligand binding ability of these membranes was dependent on the incubation temperature and corresponding tau1/2 values were 11 days at 4 degrees , 12 hr at 21 degrees , 154 min at 30 degrees and 51 min at 37 degrees , which revealed the apparent activation energy of this process to be 130+/-4 kJ/mol. Scatchard analysis of the saturation curves of [3H]CCK-8S binding was best described by a one site binding model with a Kd = 0.63+/-0.18 nM and a maximum binding of 32+/-2 fmol/mg protein. The stable GTP analogue guanosin-5'-O-(3-thiotriphosphate) (GTPgammaS) decreased the affinity of [3H]CCK-8S binding only up to 2-fold without significant influence on maximal binding. Modulation of membrane properties by different detergents revealed that only in the case of digitonin (0.03-0.04%) did the GTP-dependence of [3H]CCK-8S binding considerably increase without significant influence on the ligand binding properties in the absence of GTPgammaS. Other detergents studied (sodium cholate, sodium deoxycholate, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS), sucrose monolaurate, series Triton X and Tween) either had little influence on GTP-gammaS-dependence of [3H]CCK-8S binding or inactivated the receptor. Parallel studies of fluorescent polarization of diphenylhexatriene (DPH) in rat cortical membranes indicated that digitonin was the only detergent which at low concentrations caused a rapid increase in membrane fluidity and thereafter stabilized it at a certain level. Other detergents studied had only moderate influence on membrane fluidity (CHAPS, cholate, deoxycholate) or caused fast and continuous increase

  2. A Plasmodium falciparum copper-binding membrane protein with copper transport motifs

    PubMed Central

    2012-01-01

    Background Copper is an essential catalytic co-factor for metabolically important cellular enzymes, such as cytochrome-c oxidase. Eukaryotic cells acquire copper through a copper transport protein and distribute intracellular copper using molecular chaperones. The copper chelator, neocuproine, inhibits Plasmodium falciparum ring-to-trophozoite transition in vitro, indicating a copper requirement for malaria parasite development. How the malaria parasite acquires or secretes copper still remains to be fully elucidated. Methods PlasmoDB was searched for sequences corresponding to candidate P. falciparum copper-requiring proteins. The amino terminal domain of a putative P. falciparum copper transport protein was cloned and expressed as a maltose binding fusion protein. The copper binding ability of this protein was examined. Copper transport protein-specific anti-peptide antibodies were generated in chickens and used to establish native protein localization in P. falciparum parasites by immunofluorescence microscopy. Results Six P. falciparum copper-requiring protein orthologs and a candidate P. falciparum copper transport protein (PF14_0369), containing characteristic copper transport protein features, were identified in PlasmoDB. The recombinant amino terminal domain of the transport protein bound reduced copper in vitro and within Escherichia coli cells during recombinant expression. Immunolocalization studies tracked the copper binding protein translocating from the erythrocyte plasma membrane in early ring stage to a parasite membrane as the parasites developed to schizonts. The protein appears to be a PEXEL-negative membrane protein. Conclusion Plasmodium falciparum parasites express a native protein with copper transporter characteristics that binds copper in vitro. Localization of the protein to the erythrocyte and parasite plasma membranes could provide a mechanism for the delivery of novel anti-malarial compounds. PMID:23190769

  3. Covalent attachment of functionalized lipid bilayers to planar waveguides for measuring protein binding to biomimetic membranes.

    PubMed Central

    Heyse, S.; Vogel, H.; Sänger, M.; Sigrist, H.

    1995-01-01

    A new method is presented for measuring sensitively the interactions between ligands and their membrane-bound receptors in situ using integrated optics, thus avoiding the need for additional labels. Phospholipid bilayers were attached covalently to waveguides by a novel protocol, which can in principle be used with any glass-like surface. In a first step, phospholipids carrying head-group thiols were covalently immobilized onto SiO2-TiO2 waveguide surfaces. This was accomplished by acylation of aminated waveguides with the heterobifunctional crosslinker N-succinimidyl-3-maleimidopropionate, followed by the formation of thioethers between the surface-grafted maleimides and the synthetic thiolipids. The surface-attached thiolipids served as hydrophobic templates and anchors for the deposition of a complete lipid bilayer either by fusion of lipid vesicles or by lipid self-assembly from mixed lipid/detergent micelles. The step-by-step lipid bilayer formation on the waveguide surface was monitored in situ by an integrated optics technique, allowing the simultaneous determination of optical thickness and one of the two refractive indices of the adsorbed organic layers. Surface coverages of 50-60% were calculated for thiolipid layers. Subsequent deposition of POPC resulted in an overall lipid layer thickness of 45-50 A, which corresponds to the thickness of a fluid bilayer membrane. Specific recognition reactions occurring at cell membrane surfaces were modeled by the incorporation of lipid-anchored receptor molecules into the supported bilayer membranes. (1) The outer POPC layer was doped with biotinylated phosphatidylethanolamine. Subsequent specific binding of streptavidin was optically monitored. (2) A lipopeptide was incorporated in the outer POPC monolayer. Membrane binding of monoclonal antibodies, which were directed against the peptide moiety of the lipopeptide, was optically detected. The specific antibody binding correlated well with the lipopepitde

  4. Lipid Selectivity, Orientation, and Extent of Membrane Binding of Nonacylated RP2.

    PubMed

    Demers, Éric; Boisselier, Élodie; Horchani, Habib; Blaudez, Daniel; Calvez, Philippe; Cantin, Line; Belley, Nicolas; Champagne, Sophie; Desbat, Bernard; Salesse, Christian

    2015-04-28

    Retinitis pigmentosa 2 (RP2) is an ubiquitary protein of 350 residues. The N-terminus of RP2 contains putative sites of myristoylation and palmitoylation. The dually acylated protein is predominantly localized to the plasma membrane. However, clinically occurring substitution mutations of RP2 in photoreceptors lead to the expression of a nonacylated protein, which was shown to be misrouted to intracellular organelles using different cell lines. However, the parameters responsible for the modulation of the membrane binding of nonacylated RP2 (naRP2) are still largely unknown. The maximal insertion pressure of naRP2 has thus been determined after its injection into the subphase underneath monolayers of phospholipids, which are typical of photoreceptor membranes. These data demonstrated that naRP2 shows a preferential binding to saturated phospholipid monolayers. Moreover, polarization modulation infrared reflection absorption spectroscopy has allowed comparison of the secondary structure of this protein in solution and upon binding to phospholipid monolayers. In addition, simulations of these spectra have allowed to determine that the β-helix of naRP2 has an orientation of 60° with respect to the normal, which remains unchanged regardless of the type of phospholipid. Finally, ellipsometric measurements of naRP2 demonstrated that its particular affinity for saturated phospholipids can be explained by its larger extent of insertion in this phospholipid monolayer compared to that in polyunsaturated phospholipid monolayers. PMID:25844643

  5. Natamycin blocks fungal growth by binding specifically to ergosterol without permeabilizing the membrane.

    PubMed

    te Welscher, Yvonne M; ten Napel, Hendrik H; Balagué, Miriam Masià; Souza, Cleiton M; Riezman, Howard; de Kruijff, Ben; Breukink, Eefjan

    2008-03-01

    Natamycin is a polyene antibiotic that is commonly used as an antifungal agent because of its broad spectrum of activity and the lack of development of resistance. Other polyene antibiotics, like nystatin and filipin are known to interact with sterols, with some specificity for ergosterol thereby causing leakage of essential components and cell death. The mode of action of natamycin is unknown and is investigated in this study using different in vitro and in vivo approaches. Isothermal titration calorimetry and direct binding studies revealed that natamycin binds specifically to ergosterol present in model membranes. Yeast sterol biosynthetic mutants revealed the importance of the double bonds in the B-ring of ergosterol for the natamycin-ergosterol interaction and the consecutive block of fungal growth. Surprisingly, in strong contrast to nystatin and filipin, natamycin did not change the permeability of the yeast plasma membrane under conditions that growth was blocked. Also, in ergosterol containing model membranes, natamycin did not cause a change in bilayer permeability. This demonstrates that natamycin acts via a novel mode of action and blocks fungal growth by binding specifically to ergosterol. PMID:18165687

  6. [The effect of cocaine on binding of tricyclic antidepressives in the synaptic plasma membranes in the brain].

    PubMed

    Bures, P; Krulík, R; Fisar, Z; Fuksová, K

    1993-10-01

    Effect of cocaine on binding of 3H-imipramine, 3H-desmethylimipramine, 3H-didesmethylimipramine and 3H-amitriptyline to brain synaptic plasma membranes (SPM) was studied. Binding of methylated tricyclic antidepressants was more affected. Cocaine inhibits 3H-imipramine binding at concentrations higher than 10(-5) mol/l. Binding stimulated by phosphatidylserine was affected more significantly. PMID:8269521

  7. The polybasic region is not essential for membrane binding of the matrix protein M1 of influenza virus

    SciTech Connect

    Thaa, Bastian; Herrmann, Andreas; Veit, Michael

    2009-01-05

    The matrix protein M1, the organizer of assembly of influenza virus, interacts with other virus components and with cellular membranes. It has been proposed that M1 binding to lipids is mediated by its polybasic region, but this could hitherto not been investigated in vivo since M1 accumulates in the nucleus of transfected cells. We have equipped M1 with nuclear export signals and showed that the constructs are bound to cellular membranes. Exchange of the complete polybasic region and of further hydrophobic amino acids in its vicinity did not prevent association of M1 with membranes. We therefore suppose that M1 probably interacts with membranes via multiple binding sites.

  8. Cholesterol Modifies Huntingtin Binding to, Disruption of, and Aggregation on Lipid Membranes.

    PubMed

    Gao, Xiang; Campbell, Warren A; Chaibva, Maxmore; Jain, Pranav; Leslie, Ashley E; Frey, Shelli L; Legleiter, Justin

    2016-01-12

    Huntington's disease (HD) is an inherited neurodegenerative disease caused by abnormally long CAG-repeats in the huntingtin gene that encode an expanded polyglutamine (polyQ) domain near the N-terminus of the huntingtin (htt) protein. Expanded polyQ domains are directly correlated to disease-related htt aggregation. Htt is found highly associated with a variety of cellular and subcellular membranes that are predominantly comprised of lipids. Since cholesterol homeostasis is altered in HD, we investigated how varying cholesterol content modifies the interactions between htt and lipid membranes. A combination of Langmuir trough monolayer techniques, vesicle permeability and binding assays, and in situ atomic force microscopy were used to directly monitor the interaction of a model, synthetic htt peptide and a full-length htt-exon1 recombinant protein with model membranes comprised of total brain lipid extract (TBLE) and varying amounts of exogenously added cholesterol. As the cholesterol content of the membrane increased, the extent of htt insertion decreased. Vesicles containing extra cholesterol were resistant to htt-induced permeabilization. Morphological and mechanical changes in the bilayer associated with exposure to htt were also drastically altered by the presence of cholesterol. Disrupted regions of pure TBLE bilayers were grainy in appearance and associated with a large number of globular aggregates. In contrast, morphological changes induced by htt in bilayers enriched in cholesterol were plateau-like with a smooth appearance. Collectively, these observations suggest that the presence and amount of cholesterol in lipid membranes play a critical role in htt binding and aggregation on lipid membranes. PMID:26652744

  9. Complete dissociation of the HIV-1 gp41 ectodomain and membrane proximal regions upon phospholipid binding

    PubMed Central

    Roche, Julien; Louis, John M.; Aniana, Annie; Ghirlando, Rodolfo; Bax, Ad

    2015-01-01

    The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. Strong lipid affinity of the ectodomain suggests that its heptad repeat regions play an active role in destabilizing membranes by directly binding to the lipid bilayers and thereby lowering the free-energy barrier for membrane fusion. In such a model, immediately following the shedding of gp120, the N-heptad and C-heptad helices dissociate and melt into the host cell and viral membranes, respectively, pulling the destabilized membranes into juxtaposition, ready for fusion. Post-fusion, reaching the final 6-helix bundle (6HB) conformation then involves competition between intermolecular interactions needed for formation of the symmetric 6HB trimer and the membrane affinity of gp41's ectodomain, including its membrane-proximal regions. Our solution NMR study of the structural and dynamic properties of three constructs containing the ectodomain of gp41 with and without its membrane-proximal regions suggests that these segments do not form inter-helical interactions until the very late steps of the fusion process. Interactions between the polar termini of the heptad regions, which are not associating with the lipid surface, therefore may constitute the main driving force initiating formation of the final post-fusion states. The absence of significant intermolecular ectodomain interactions in the presence of dodecyl phosphocholine highlights the importance of trimerization of gp41’s transmembrane helix to prevent complete dissociation of the trimer during the course of fusion. PMID:25631354

  10. Ovarian overproduction of androgens

    MedlinePlus

    ... syndrome (PCOS) can both cause too much androgen production. Cushing's disease is a problem with the pituitary ... the adrenal glands can also cause too much production of androgens and can lead to male body ...

  11. Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM

    NASA Astrophysics Data System (ADS)

    Pfreundschuh, Moritz; Alsteens, David; Wieneke, Ralph; Zhang, Cheng; Coughlin, Shaun R.; Tampé, Robert; Kobilka, Brian K.; Müller, Daniel J.

    2015-11-01

    A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of suitable nanoscopic methods. Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands. Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1. We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s. Surface chemistry and nanoscopic method are applicable to a range of biological systems in vitro and in vivo and to concurrently detect and localize multiple ligand-binding sites at single receptor resolution.

  12. Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM

    PubMed Central

    Pfreundschuh, Moritz; Alsteens, David; Wieneke, Ralph; Zhang, Cheng; Coughlin, Shaun R.; Tampé, Robert; Kobilka, Brian K.; Müller, Daniel J.

    2015-01-01

    A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of suitable nanoscopic methods. Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands. Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1. We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s. Surface chemistry and nanoscopic method are applicable to a range of biological systems in vitro and in vivo and to concurrently detect and localize multiple ligand-binding sites at single receptor resolution. PMID:26561004

  13. Membrane proteins specified by herpes simplex viruses. V. Identification of an Fc-binding glycoprotein.

    PubMed Central

    Baucke, R B; Spear, P G

    1979-01-01

    A glycoprotein with affinity for the Fc region of immunoglobulin was isolated from extracts of cultured cells infected with herpes simplex virus type 1, and experiments were done to characterize its properties and to investigate whether it could account for the Fc-binding activity previously demonstrated on the surfaces of intact herpes simplex virus-infected cells. The technique of affinity chromatography was used to identify and isolate the Fc-binding glycoprotein and to demonstrate the specificity of its interaction with immunoglobulin G-Fc. Although three electrophoretically distinguishable Fc-binding polypeptides were identified by affinity chromatography, these three species appear to be different forms of the same translation product, based on comparisons of proteolytic digestion products and on the kinetics of appearance of each form after a brief pulse with radioactive amino acids. The results suggest that one polypeptide, designated pE, is processed to yield gE1, which is in turn processed to yield gE2. Both gE1 and gE2 are glycosylated membrane proteins and both can be labeled by the lactoperoxidase-catalyzed radioiodination of intact infected cells, indicating the presence of these proteins in surface membranes of the cells. Increases in the amounts of gE1 and gE2 at the cell surface were found to parallel the increase in Fc-binding activity of intact infected cells. Images PMID:229267

  14. Calcium Binding by Ro 60 Multiple Antigenic Peptides on PVDF Membrane.

    PubMed

    Kurien, Biji T; Bachmann, Michael P

    2015-01-01

    Antibodies directed against ribonucleoprotein (RNP) particles are observed in systemic lupus erythematosus. Ro RNP particle is one such target. It is composed of a 60 kDa protein (Ro 60 or SS-A) that is non-covalently associated with at least one of the four short uridine-rich RNAs (the hY RNAs). Previously, we showed that multiple antigenic peptides (MAPs) made from the sequence of the Ro 60 autoantigen could be used, using double-immunodiffusion studies, enzyme-linked immunosorbant assay, affinity chromatography, and surface plasmon resonance, to show intramolecular and intermolecular protein-protein interaction within the Ro 60 RNP particle. We also observed that calcium is important in mediating this interaction. We hypothesized, therefore, that 60 kDa Ro is a calcium-binding protein. To investigate this, we electrophoresed 60 kDa Ro MAPs, transferred them to PVDF membrane, and assayed calcium binding using the Quin-2 system. Several Ro 60 MAPs were found to bind calcium using this assay, as well as bovine serum albumin, another calcium-binding protein. However, a MAP constructed from the Sm autoantigen did not bind to calcium. These data, along with our observation regarding the involvement of calcium in protein-protein interaction occurring between Ro 60 antigen and Ro 60 MAPs, makes us propose that Ro 60 antigen is a calcium-binding protein. PMID:26139264

  15. Pathogenesis of Shigella diarrhea: rabbit intestinal cell microvillus membrane binding site for Shigella toxin

    SciTech Connect

    Fuchs, G.; Mobassaleh, M.; Donohue-Rolfe, A.; Montgomery, R.K.; Grand, R.J.; Keusch, G.T.

    1986-08-01

    This study examined the binding of purified /sup 125/I-labeled shigella toxin to rabbit jejunal microvillus membranes (MVMs). Toxin binding was concentration dependent, saturable, reversible, and specifically inhibited by unlabeled toxin. The calculated number of toxin molecules bound at 4/sup 0/C was 7.9 X 10(10) (3 X 10(10) to 2 X 10(11))/micrograms of MVM protein or 1.2 X 10(6) per enterocyte. Scatchard analysis showed the binding site to be of a single class with an equilibrium association constant, K, of 4.7 X 10(9) M-1 at 4/sup 0/C. Binding was inversely related to the temperature of incubation. A total of 80% of the labeled toxin binding at 4/sup 0/C dissociated from MVM when the temperature was raised to 37/sup 0/C, but reassociated when the temperature was again brought to 4/sup 0/C. There was no structural or functional change of MVM due to toxin as monitored by electron microscopy or assay of MVM sucrase activity. These studies demonstrate a specific binding site for shigella toxin on rabbit MVMs. The physiological relevance of this receptor remains to be determined.

  16. Membrane folate-binding proteins are responsible for folate-protein conjugate endocytosis into cultured cells.

    PubMed Central

    Leamon, C P; Low, P S

    1993-01-01

    Folate-protein conjugates have been shown to bind to and enter HeLa and KB cells by receptor-mediated endocytosis [Leamon and Low (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5572-5576]. Although these cells contain a membrane folate-binding protein (FBP) involved in the uptake of free folate, no studies have been conducted to evaluate whether the folate-protein conjugates enter cells via the same protein. To address this issue, HeLa cell monolayers were treated with folate-labelled 125I-RNAase under various conditions characteristic of FBP-mediated folate uptake. Folate-labelled 125I-RNAase was found to bind to cells with high affinity (Kd = 24 nM), and like the free vitamin, its binding could be competitively blocked by excess free folate. Furthermore, binding could be reversed by either washing the cells with acid/saline, pH 3.0, or by treating the cells with phosphatidyl-inositol-specific phospholipase C, an enzyme known to release FBP from cell surfaces. Because cells pretreated with anti-FBP serum were unable to bind folate conjugates, and since the same antiserum identified a single 65 kDa band reminiscent of FBPs found in many other tissues, we conclude that a classical FBP is responsible for the uptake of folate-protein conjugates by receptor-bearing cells. Images Figure 5 PMID:8387781

  17. Sulfate inhibits ( sup 14 C)phosphonoformic acid binding to renal brush-border membranes

    SciTech Connect

    Tenenhouse, H.S.; Lee, J. )

    1990-08-01

    To examine the specificity of the phosphonoformic acid (PFA) interaction with the Na(+)-dependent phosphate transporter of mouse renal brush-border membrane vesicles, we compared the effects of anions on Na(+)-dependent (14C)PFA binding and Na(+)-dependent phosphate transport. Inhibition of PFA binding was achieved by PFA (% control = 0 +/- 1), sulfate (15 +/- 2), arsenate (35 +/- 1), phosphate (59 +/- 2), and nitrate (68 +/- 4), whereas inhibition of phosphate transport was only apparent with phosphate (0 +/- 1), PFA (22 +/- 4), and arsenate (37 +/- 5). Succinate and gluconate had no effect on either Na(+)-dependent process. Under conditions where Na(+)-dependent PFA binding was maximally inhibited by phosphate (% control = 65 +/- 4), further inhibition could be achieved by sulfate (26 +/- 5%). Na(+)-dependent PFA binding was competitively inhibited by phosphate (apparent Ki = 8.9 +/- 1.2 mM) and noncompetitively inhibited by sulfate (apparent Ki = 2.6 +/- 0.5 mM). We found that PFA inhibited Na(+)-dependent sulfate transport (50% inhibition at 9 mM PFA) as well as Na(+)-dependent phosphate transport (50% inhibition at 0.5 mM PFA). We also examined the pH dependence of Na(+)-dependent PFA binding and Na(+)-dependent phosphate and sulfate transport. PFA binding was optimal at pH = 7.4, whereas phosphate transport increased with increasing pH, and sulfate transport increased with decreasing pH.

  18. Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells

    PubMed Central

    Wang, Wei; Yang, Yunze; Wang, Shaopeng; Nagaraj, Vinay J; Liu, Qiang; Wu, Jie; Tao, Nongjian

    2013-01-01

    Membrane proteins (MPs) mediate a variety of cellular responses to extracellular signals. While MPs are intensely studied for their values as disease biomarkers and therapeutic targets, in situ investigation of binding kinetics of MPs with their ligands has been a challenge. Traditional approaches isolate MPs and then study them ex situ, which does not accurately reflect their native structures and functions. We present here a label-free plasmonic microscopy method to map the local binding kinetics of MPs in their native environment. This new analytical method can perform simultaneous plasmonic and fluorescence imaging, thus making it possible to combine the strengths of both label-based and label-free techniques in one system. Using this method, we have determined the distribution of MPs on the surface of single cells, and the local binding kinetic constants of different MPs. Furthermore, we have studied the polarization of the MPs on the cell surface during chemotaxis. PMID:23000999

  19. GLP-1(7-36)amide binding in skeletal muscle membranes from streptozotocin diabetic rats.

    PubMed

    Villanueva-Peñacarrillo, M L; Delgado, E; Vicent, D; Mérida, E; Alcántara, A I; Valverde, I

    1995-09-01

    A higher specific binding of GLP-1(7-36)amide is found in skeletal muscle plasma membranes from adult streptozotocin (STZ)-treated rats (insulin-dependent diabetes mellitus model) and from neonatal STZ-treated rats (non insulin-dependent diabetes mellitus model), as compared to that in normal controls; no apparent change in the affinity was observed, that indicating the presence in both diabetic models of an increased number of high affinity binding sites for the peptide. The maximal specific GLP-1(7-16)amide binding in the non insulin-dependent diabetes mellitus model was found to be significantly higher than that in the insulin-dependent diabetes mellitus model. As GLP-1(7-36)amide exerts a glycogenic effect in the rat skeletal muscle, the present data suggest that the action of the peptide in the muscle glucose metabolism may be increased in states of insulin deficiency accompanied or not by insulin resistance. PMID:21153227

  20. Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain

    PubMed Central

    Barros, Marilia; Jin, Danni; Lösche, Mathias; Vogt, Volker M.

    2015-01-01

    ABSTRACT The principles underlying membrane binding and assembly of retroviral Gag proteins into a lattice are understood. However, little is known about how these processes are related. Using purified Rous sarcoma virus Gag and Gag truncations, we studied the interrelation of Gag-Gag interaction and Gag-membrane interaction. Both by liposome binding and by surface plasmon resonance on a supported bilayer, Gag bound to membranes much more tightly than did matrix (MA), the isolated membrane binding domain. In principle, this difference could be explained either by protein-protein interactions leading to cooperativity in membrane binding or by the simultaneous interaction of the N-terminal MA and the C-terminal nucleocapsid (NC) of Gag with the bilayer, since both are highly basic. However, we found that NC was not required for strong membrane binding. Instead, the spacer peptide assembly domain (SPA), a putative 24-residue helical sequence comprising the 12-residue SP segment of Gag and overlapping the capsid (CA) C terminus and the NC N terminus, was required. SPA is known to be critical for proper assembly of the immature Gag lattice. A single amino acid mutation in SPA that abrogates assembly in vitro dramatically reduced binding of Gag to liposomes. In vivo, plasma membrane localization was dependent on SPA. Disulfide cross-linking based on ectopic Cys residues showed that the contacts between Gag proteins on the membrane are similar to the known contacts in virus-like particles. Taken together, we interpret these results to mean that Gag membrane interaction is cooperative in that it depends on the ability of Gag to multimerize. IMPORTANCE The retroviral structural protein Gag has three major domains. The N-terminal MA domain interacts directly with the plasma membrane (PM) of cells. The central CA domain, together with immediately adjoining sequences, facilitates the assembly of thousands of Gag molecules into a lattice. The C-terminal NC domain interacts with

  1. Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer’s-Associated Aβ Oligomers

    PubMed Central

    Wilcox, Kyle C.; Marunde, Matthew R.; Das, Aditi; Velasco, Pauline T.; Kuhns, Benjamin D.; Marty, Michael T.; Jiang, Haoming; Luan, Chi-Hao; Sligar, Stephen G.; Klein, William L.

    2015-01-01

    Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS) tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs). AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer’s dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs). This method gives a soluble membrane protein library (SMPL)—a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer’s model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can facilitate drug

  2. Lipid-Free Antigen B Subunits from Echinococcus granulosus: Oligomerization, Ligand Binding, and Membrane Interaction Properties

    PubMed Central

    Silva-Álvarez, Valeria; Franchini, Gisela R.; Pórfido, Jorge L.; Kennedy, Malcolm W.; Ferreira, Ana M.; Córsico, Betina

    2015-01-01

    Background The hydatid disease parasite Echinococcus granulosus has a restricted lipid metabolism, and needs to harvest essential lipids from the host. Antigen B (EgAgB), an abundant lipoprotein of the larval stage (hydatid cyst), is thought to be important in lipid storage and transport. It contains a wide variety of lipid classes, from highly hydrophobic compounds to phospholipids. Its protein component belongs to the cestode-specific Hydrophobic Ligand Binding Protein family, which includes five 8-kDa isoforms encoded by a multigene family (EgAgB1-EgAgB5). How lipid and protein components are assembled into EgAgB particles remains unknown. EgAgB apolipoproteins self-associate into large oligomers, but the functional contribution of lipids to oligomerization is uncertain. Furthermore, binding of fatty acids to some EgAgB subunits has been reported, but their ability to bind other lipids and transfer them to acceptor membranes has not been studied. Methodology/Principal Findings Lipid-free EgAgB subunits obtained by reverse-phase HPLC were used to analyse their oligomerization, ligand binding and membrane interaction properties. Size exclusion chromatography and cross-linking experiments showed that EgAgB8/2 and EgAgB8/3 can self-associate, suggesting that lipids are not required for oligomerization. Furthermore, using fluorescent probes, both subunits were found to bind fatty acids, but not cholesterol analogues. Analysis of fatty acid transfer to phospholipid vesicles demonstrated that EgAgB8/2 and EgAgB8/3 are potentially capable of transferring fatty acids to membranes, and that the efficiency of transfer is dependent on the surface charge of the vesicles. Conclusions/Significance We show that EgAgB apolipoproteins can oligomerize in the absence of lipids, and can bind and transfer fatty acids to phospholipid membranes. Since imported fatty acids are essential for Echinococcus granulosus, these findings provide a mechanism whereby EgAgB could engage in lipid

  3. Proteins of rough microsomal membranes related to ribosome binding. II. Cross-linking of bound ribosomes to specific membrane proteins exposed at the binding sites

    PubMed Central

    1978-01-01

    Two proteins (ribophorins I and II), which are integral components of rough microsomal membranes and appear to be related to the bound ribosomes, were shown to be exposed on the surface of rat liver rough microsomes (RM) and to be in close proximity to the bound ribosomes. Both proteins were labeled when intact RM were incubated with a lactoperoxidase iodinating system, but only ribophorin I was digested during mild trypsinization of intact RM. Ribophorin II (63,000 daltons) was only proteolyzed when the luminal face of the microsomal vesicles was made accessible to trypsin by the addition of sublytical detergent concentrations. Only 30--40% of the bound ribosomes were released during trypsinization on intact RM, but ribosome release was almost complete in the presence of low detergent concentrations. Very low glutaraldehyde concentrations (0.005--0.02%) led to the preferential cross-linking of large ribosomal subunits of bound ribosomes to the microsomal membranes. This cross-linking prevented the release of subunits caused by puromycin in media of high ionic strength, but not the incorporation of [3H]puromycin into nascent polypeptide chains. SDS- acrylamide gel electrophoresis of cross-linked samples a preferential reduction in the intensity of the bands representing the ribophorins and the formation of aggregates which did not penetrate into the gels. At low methyl-4-mercaptobutyrimidate (MMB) concentrations (0.26 mg/ml) only 30% of the ribosomes were cross-linked to the microsomal membranes, as shown by the puromycin-KCl test, but membranes could still be solubilized with 1% DOC. This allowed the isolation of the ribophorins together with the sedimentable ribosomes, as was shown by electrophoresis of the sediments after disruption of the cross-links by reduction. Experiments with RM which contained only inactive ribosomes showed that the presence of nascent chains was not necessary for the reversible cross-linking of ribosomes to the membranes. These

  4. Binding of amphiphilic and triphilic block copolymers to lipid model membranes: the role of perfluorinated moieties.

    PubMed

    Schwieger, Christian; Achilles, Anja; Scholz, Sven; Rüger, Jan; Bacia, Kirsten; Saalwaechter, Kay; Kressler, Jörg; Blume, Alfred

    2014-09-01

    A novel class of symmetric amphi- and triphilic (hydrophilic, lipophilic, fluorophilic) block copolymers has been investigated with respect to their interactions with lipid membranes. The amphiphilic triblock copolymer has the structure PGMA(20)-PPO(34)-PGMA(20) (GP) and it becomes triphilic after attaching perfluoroalkyl moieties (F9) to either end which leads to F(9)-PGMA(20)-PPO(34)-PGMA(20)-F(9) (F-GP). The hydrophobic poly(propylene oxide) (PPO) block is sufficiently long to span a lipid bilayer. The poly(glycerol monomethacrylate) (PGMA) blocks have a high propensity for hydrogen bonding. The hydrophobic and lipophobic perfluoroalkyl moieties have the tendency to phase segregate in aqueous as well as in hydrocarbon environments. We performed differential scanning calorimetry (DSC) measurements on polymer bound lipid vesicles under systematic variation of the bilayer thickness, the nature of the lipid headgroup, and the polymer concentration. The vesicles were composed of phosphatidylcholines (DMPC, DPPC, DAPC, DSPC) or phosphatidylethanolamines (DMPE, DPPE, POPE). We showed that GP as well as F-GP binding have membrane stabilizing and destabilizing components. PPO and F9 blocks insert into the hydrophobic part of the membrane concomitantly with PGMA block adsorption to the lipid headgroup layer. The F9 chains act as additional membrane anchors. The insertion of the PPO blocks of both GP and F-GP could be proven by 2D-NOESY NMR spectroscopy. By fluorescence microscopy we show that F-GP binding increases the porosity of POPC giant unilamellar vesicles (GUVs), allowing the influx of water soluble dyes as well as the translocation of the complete triphilic polymer and its accumulation at the GUV surface. These results open a new route for the rational design of membrane systems with specific properties. PMID:24942348

  5. Characterisation of Conformational and Ligand Binding Properties of Membrane Proteins Using Synchrotron Radiation Circular Dichroism (SRCD).

    PubMed

    Hussain, Rohanah; Siligardi, Giuliano

    2016-01-01

    Membrane proteins are notoriously difficult to crystallise for use in X-ray crystallographic structural determination, or too complex for NMR structural studies. Circular dichroism (CD) is a fast and relatively easy spectroscopic technique to study protein conformational behaviour in solution. The advantage of synchrotron radiation circular dichroism (SRCD) measured with synchrotron beamlines compared to the CD from benchtop instruments is the extended spectral far-UV region that increases the accuracy of secondary structure estimations, in particular under high ionic strength conditions. Membrane proteins are often available in small quantities, and for this SRCD measured at the Diamond B23 beamline has successfully facilitated molecular recognition studies. This was done by probing the local tertiary structure of aromatic amino acid residues upon addition of chiral or non-chiral ligands using long pathlength cells (1-5 cm) of small volume capacity (70 μl-350 μl). In this chapter we describe the use of SRCD to qualitatively and quantitatively screen ligand binding interactions (exemplified by Sbma, Ace1 and FsrC proteins); to distinguish between functionally similar drugs that exhibit different mechanisms of action towards membrane proteins (exemplified by FsrC); and to identify suitable detergent conditions to observe membrane protein-ligand interactions using stabilised proteins (exemplified by inositol transporters) as well as the stability of membrane proteins (exemplified by GalP, Ace1). The importance of the in solution characterisation of the conformational behaviour and ligand binding properties of proteins in both far- andnear-UV regions and the use of high-throughput CD (HT-CD) using 96- and 384-well multiplates to study the folding effects in various protein crystallisation buffers are also discussed. PMID:27553234

  6. Interplay of electrostatics and lipid packing determines the binding of charged polymer coated nanoparticles to model membranes.

    PubMed

    Biswas, Nupur; Bhattacharya, Rupak; Saha, Arindam; Jana, Nikhil R; Basu, Jaydeep K

    2015-10-01

    Understanding of nanoparticle-membrane interactions is useful for various applications of nanoparticles like drug delivery and imaging. Here we report on the studies of interaction between hydrophilic charged polymer coated semiconductor quantum dot nanoparticles with model lipid membranes. Atomic force microscopy and X-ray reflectivity measurements suggest that cationic nanoparticles bind and penetrate bilayers of zwitterionic lipids. Penetration and binding depend on the extent of lipid packing and result in the disruption of the lipid bilayer accompanied by enhanced lipid diffusion. On the other hand, anionic nanoparticles show minimal membrane binding although, curiously, their interaction leads to reduction in lipid diffusivity. It is suggested that the enhanced binding of cationic QDs at higher lipid packing can be understood in terms of the effective surface potential of the bilayers which is tunable through membrane lipid packing. Our results bring forth the subtle interplay of membrane lipid packing and electrostatics which determine nanoparticle binding and penetration of model membranes with further implications for real cell membranes. PMID:26327393

  7. Classical Conditioning of the Rabbit Eyelid Response Increases Glutamate Receptor Binding in Hippocampal Synaptic Membranes

    NASA Astrophysics Data System (ADS)

    Mamounas, Laura A.; Thompson, Richard F.; Lynch, Gary; Baudry, Michel

    1984-04-01

    Hippocampal pyramidal neurons exhibit a rapid within-trial increase in firing frequency during classical conditioning of the rabbit eyelid response. It has been proposed that the cellular mechanisms responsible for hippocampal long-term potentiation (LTP) may also mediate this learning-dependent increase in neuronal activity. The induction of LTP in rat hippocampal slices results in an increase in the number of [3H]glutamate-binding sites in the potentiated region. The present study investigates the kinetics of [3H]glutamate binding to hippocampal synaptic membranes after eyelid conditioning in the rabbit. We report that the regional distribution of [3H]glutamate binding across the layers of rabbit hippocampus is compatible with a dendritic localization. The pharmacological and ionic properties of the binding suggest that it is associated with an excitatory amino acid receptor. After eyelid conditioning, the maximal number of hippocampal [3H]glutamate-binding sites is increased in animals receiving paired presentations of the tone conditioned stimulus and corneal air-puff unconditioned stimulus relative to that found in naive or unpaired control animals. These results strengthen the hypothesis that an LTP-like mechanism underlies the increase in hippocampal firing frequency during rabbit eyelid conditioning.

  8. Characterization of GTP binding and hydrolysis in plasma membranes of zucchini

    NASA Technical Reports Server (NTRS)

    Perdue, D. O.; Lomax, T. L.

    1992-01-01

    We have investigated the possibility that G-protein-like entities may be present in the plasma membrane (PM) of zucchini (Cucurbita pepo L.) hypocotyls by examining a number of criteria common to animal and yeast G-proteins. The GTP binding and hydrolysis characteristics of purified zucchini PM are similar to the characteristics of a number of known G-proteins. Our results demonstrate GTP binding to a single PM site having a Kd value between 16-31 nM. This binding has a high specificity for guanine nucleotides, and is stimulated by Mg2+, detergents, and fluoride or aluminium ions. The GTPase activity (Km = 0.49 micromole) of zucchini PM shows a sensitivity to NaF similar to that seen for other G-proteins. Localization of GTP mu 35S binding to nitrocellulose blots of proteins separated by SDS-PAGE indicates a 30-kDa protein as the predominant GTP-binding species in zucchini PM. Taken together, these data indicate that plant PM contains proteins which are biochemically similar to previously characterized G-proteins.

  9. Tension-compression asymmetry in the binding affinity of membrane-anchored receptors and ligands

    NASA Astrophysics Data System (ADS)

    Xu, Guang-Kui; Liu, Zishun; Feng, Xi-Qiao; Gao, Huajian

    2016-03-01

    Cell adhesion plays a crucial role in many biological processes of cells, e.g., immune responses, tissue morphogenesis, and stem cell differentiation. An essential problem in the molecular mechanism of cell adhesion is to characterize the binding affinity of membrane-anchored receptors and ligands under different physiological conditions. In this paper, a theoretical model is presented to study the binding affinity between a large number of anchored receptors and ligands under both tensile and compressive stresses, and corroborated by demonstrating excellent agreement with Monte Carlo simulations. It is shown that the binding affinity becomes lower as the magnitude of the applied stress increases, and drops to zero at a critical tensile or compressive stress. Interestingly, the critical compressive stress is found to be substantially smaller than the critical tensile stress for relatively long and flexible receptor-ligand complexes. This counterintuitive finding is explained by using the Euler instability theory of slender columns under compression. The tension-compression asymmetry in the binding affinity of anchored receptors and ligands depends subtly on the competition between the breaking and instability of their complexes. This study helps in understanding the role of mechanical forces in cell adhesion mediated by specific binding molecules.

  10. The N-Terminal Domain of Bcl-xL Reversibly Binds Membranes in a pH-Dependent Manner†

    PubMed Central

    Thuduppathy, Guruvasuthevan R.; Terrones, Oihana; Craig, Jeffrey W.; Basañez, Gorka; Hill, R. Blake

    2006-01-01

    Bcl-xL regulates apoptosis by maintaining the integrity of the mitochondrial outer membrane by adopting both soluble and membrane-associated forms. The membrane-associated conformation does not require a conserved, C-terminal transmembrane domain and appears to be inserted into the bilayer of synthetic membranes as assessed by membrane permeabilization and critical surface pressure measurements. Membrane association is reversible and is regulated by the cooperative binding of approximately two protons to the protein. Two acidic residues, Glu153 and Asp156, that lie in a conserved hairpin of Bcl-xLΔTM appear to be important in this process on the basis of a 16% increase in the level of membrane association of the double mutant E153Q/D156N. Contrary to that for the wild type, membrane permeabilization for the mutant is not correlated with membrane association. Monolayer surface pressure measurements suggest that this effect is primarily due to less membrane penetration. These results suggest that E153 and D156 are important for the Bcl-xLΔTM conformational change and that membrane binding can be distinct from membrane permeabilization. Taken together, these studies support a model in which Bcl-xL activity is controlled by reversible insertion of its N-terminal domain into the mitochondrial outer membrane. Future studies with Bcl-xL mutants such as E153Q/D156N should allow determination of the relative contributions of membrane binding, insertion, and permeabilization to the regulation of apoptosis. PMID:17128992

  11. The Adipophilin C Terminus Is a Self-folding Membrane-binding Domain That Is Important for Milk Lipid Secretion*

    PubMed Central

    Chong, Brandi M.; Russell, Tanya D.; Schaack, Jerome; Orlicky, David J.; Reigan, Philip; Ladinsky, Mark; McManaman, James L.

    2011-01-01

    Cytoplasmic lipid droplets (CLD) in mammary epithelial cells undergo secretion by a unique membrane envelopment process to produce milk lipids. Adipophilin (ADPH/Plin2), a member of the perilipin/PAT family of lipid droplet-associated proteins, is hypothesized to mediate CLD secretion through interactions with apical plasma membrane elements. We found that the secretion of CLD coated by truncated ADPH lacking the C-terminal region encoding a putative four-helix bundle structure was impaired relative to that of CLD coated by full-length ADPH. We used homology modeling and analyses of the solution and membrane binding properties of purified recombinant ADPH C terminus to understand how this region possibly mediates CLD secretion. Homology modeling supports the concept that the ADPH C terminus forms a four-helix bundle motif and suggests that this structure can form stable membrane bilayer interactions. Circular dichroism and protease mapping studies confirmed that the ADPH C terminus is an independently folding α-helical structure that is relatively resistant to urea denaturation. Liposome binding studies showed that the purified C terminus binds to phospholipid membranes through electrostatic dependent interactions, and cell culture studies documented that it localizes to the plasma membrane. Collectively, these data provide direct evidence that the ADPH C terminus forms a stable membrane binding helical structure that is important for CLD secretion. We speculate that interactions between the four-helix bundle of ADPH and membrane phospholipids may be an initial step in milk lipid secretion. PMID:21383012

  12. Membrane modulates affinity for calcium ion to create an apparent cooperative binding response by annexin a5.

    PubMed

    Gauer, Jacob W; Knutson, Kristofer J; Jaworski, Samantha R; Rice, Anne M; Rannikko, Anika M; Lentz, Barry R; Hinderliter, Anne

    2013-06-01

    Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca²⁺) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca²⁺ in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca²⁺ differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca²⁺ affinity to a membrane-associated, higher Ca²⁺ affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca²⁺ influx is the basis for the cooperative response of annexin a5 toward Ca²⁺, and the role of membrane organization in this response. PMID:23746516

  13. Trp[superscript 2313]-His[superscript 2315] of Factor VIII C2 Domain Is Involved in Membrane Binding Structure of a Complex Between the C[subscript 2] Domain and an Inhibitor of Membrane Binding

    SciTech Connect

    Liu, Zhuo; Lin, Lin; Yuan, Cai; Nicolaes, Gerry A.F.; Chen, Liqing; Meehan, Edward J.; Furie, Bruce; Furie, Barbara; Huang, Mingdong

    2010-11-03

    Factor VIII (FVIII) plays a critical role in blood coagulation by forming the tenase complex with factor IXa and calcium ions on a membrane surface containing negatively charged phospholipids. The tenase complex activates factor X during blood coagulation. The carboxyl-terminal C2 domain of FVIII is the main membrane-binding and von Willebrand factor-binding region of the protein. Mutations of FVIII cause hemophilia A, whereas elevation of FVIII activity is a risk factor for thromboembolic diseases. The C2 domain-membrane interaction has been proposed as a target of intervention for regulation of blood coagulation. A number of molecules that interrupt FVIII or factor V (FV) binding to cell membranes have been identified through high throughput screening or structure-based design. We report crystal structures of the FVIII C2 domain under three new crystallization conditions, and a high resolution (1.15 {angstrom}) crystal structure of the FVIII C2 domain bound to a small molecular inhibitor. The latter structure shows that the inhibitor binds to the surface of an exposed {beta}-strand of the C2 domain, Trp{sup 2313}-His{sup 2315}. This result indicates that the Trp{sup 2313}-His{sup 2315} segment is an important constituent of the membrane-binding motif and provides a model to understand the molecular mechanism of the C2 domain membrane interaction.

  14. The vitellogenin cDNA of Cherax quadricarinatus encodes a lipoprotein with calcium binding ability, and its expression is induced following the removal of the androgenic gland in a sexually plastic system.

    PubMed

    Abdu, Uri; Davis, Claytus; Khalaila, Isam; Sagi, Amir

    2002-07-01

    Oocyte maturation in decapod crustaceans is a two step process. Primary vitellogenesis is followed by a variable hiatus that lasts up to the onset of secondary vitellogenesis, which is marked by the rapid accumulation of yolk proteins in the oocytes. We have cloned a complete Cherax quadricarinatus vitellogenin cDNA. The sequenced cDNA contains a 2584 aa open reading frame which shows sequence similarity to vitellogenins from other crustaceans. The mRNA encodes at least two of the previously identified vitellin components, indicating that the primary translation product is subject to post-translational modification, including proteolytic cleavage. The region close to the 3(') end of the mRNA encodes a previously characterized negatively charged protein (provisionally designated P(106)). We show here that the negative charge of P(106) could be due to its ability to bind calcium. Northern blot data show that this gene is expressed as a single 8000 nt transcript and is present in the hepatopancreas of secondary-vitellogenic females. Primary vitellogenic and other tissues examined in male and female animals were negative. In sexually plastic intersex animals, removal of the androgenic gland results in vitellogenin transcription, indicating that the gene is negatively regulated by the androgenic gland. PMID:12225768

  15. Isolation and identification of L-dopa decarboxylase as a protein that binds to and enhances transcriptional activity of the androgen receptor using the repressed transactivator yeast two-hybrid system.

    PubMed Central

    Wafa, Latif A; Cheng, Helen; Rao, Mira A; Nelson, Colleen C; Cox, Michael; Hirst, Martin; Sadowski, Ivan; Rennie, Paul S

    2003-01-01

    The AR (androgen receptor) is a ligand-regulated transcription factor, which belongs to the steroid receptor family and plays an essential role in growth and development of the prostate. Transcriptional activity of steroid receptors is modulated by interaction with co-regulator proteins and yeast two-hybrid analysis is commonly used to identify these steroid receptor-interacting proteins. However, a limitation of conventional two-hybrid systems for detecting AR protein partners has been that they only allow for analysis of the ligand- and DNA-binding domains of the receptor, as its NTD (N-terminal domain) possesses intrinsic transactivation activity. To identify AR N-terminus-interacting proteins, its NTD was used in the RTA (repressed transactivator) system, which is specifically designed for transactivator bait proteins and was shown to be suitable for two-hybrid analysis with the AR NTD. DDC (L-dopa decarboxylase) was detected multiple times as a novel AR-interacting protein, which was subsequently confirmed in vitro and in vivo. Furthermore, transient transfection of DDC in prostate cancer cells strongly enhanced ligand-dependent AR transcriptional activity, an effect that was antagonized using high concentrations of the anti-androgen bicalutamide. Glucocorticoid receptor activity was also strongly enhanced with DDC co-transfection, while oestrogen receptor activity was only mildly affected. Together, our data demonstrate that DDC interacts with AR to enhance steroid receptor transactivation, which may have important implications in prostate cancer progression. PMID:12864730

  16. Binding of [3H]-muscimol, a potent gamma-aminobutyric acid receptor agonist, to membranes of the bovine retina.

    PubMed Central

    Osborne, N. N.

    1980-01-01

    1 The binding of [3H]-muscimol, a potent gamma-aminobutyric acid (GABA) receptor agonist, to crude membrane preparations of bovine retina was studied, using a filtration method to isolate membrane-bound ligand. 2 Specific binding was found to be saturable and occurred at two binding sites with affinity constants of 4.3 nM and 38.2 nM. 3 Binding was sodium-independent, enhanced by both freezing and Triton X-100 treatment but abolished with sodium laurylsulphate. 4 The binding sites demonstrated a high degree of pharmacological specificity, GABA being a potent displacer of [3H]-muscimol. 5 A higher degree of [3H]-muscimol binding was associated with subcellular fractions enriched with photoreceptor synaptosomes rather than with fractions enriched with conventional synaptosomes. PMID:7470740

  17. Membrane binding and bending in Ebola VP40 assembly and egress

    PubMed Central

    Stahelin, Robert V.

    2014-01-01

    Lipid-enveloped viruses contain a lipid bilayer coat that protects their genome and helps to facilitate entry into the host cell. Filoviruses are lipid-enveloped viruses that have up to 90% clinical fatality and include Marbug (MARV) and Ebola (EBOV). These pleomorphic filamentous viruses enter the host cell through their membrane-embedded glycoprotein and then replicate using just seven genes encoded in their negative-sense RNA genome. EBOV budding occurs from the inner leaflet of the plasma membrane (PM) and is driven by the matrix protein VP40, which is the most abundantly expressed protein of the virus. VP40 expressed in mammalian cells alone can trigger budding of filamentous virus-like particles (VLPs) that are nearly indistinguishable from authentic EBOV. VP40, such as matrix proteins from other viruses, has been shown to bind anionic lipid membranes. However, how VP40 selectively interacts with the inner leaflet of the PM and assembles into a filamentous lipid enveloped particle is mostly unknown. This article describes what is known regarding VP40 membrane interactions and what answers will fill the gaps. PMID:24995005

  18. Diffusion coefficients and dissociation constants of enhanced green fluorescent protein binding to free standing membranes.

    PubMed

    Thomas, Franziska A; Visco, Ilaria; Petrášek, Zdeněk; Heinemann, Fabian; Schwille, Petra

    2015-12-01

    Recently, a new and versatile assay to determine the partitioning coefficient [Formula: see text] as a measure for the affinity of peripheral membrane proteins for lipid bilayers was presented in the research article entitled, "Introducing a fluorescence-based standard to quantify protein partitioning into membranes" [1]. Here, the well-characterized binding of hexahistidine-tag (His6) to NTA(Ni) was utilized. Complementarily, this data article reports the average diffusion coefficient [Formula: see text] of His6-tagged enhanced green fluorescent protein (eGFP-His6) and the fluorescent lipid analog ATTO-647N-DOPE in giant unilamellar vesicles (GUVs) containing different amounts of NTA(Ni) lipids. In addition, dissociation constants [Formula: see text] of the NTA(Ni)/eGFP-His6 system are reported. Further, a conversion between [Formula: see text] and [Formula: see text] is provided. PMID:26587560

  19. Removal of arsenic from groundwater using reagent binding/membrane separation

    SciTech Connect

    Legault, A.S.; Volchek, K.; Whittaker, H. ); Tremblay, A.Y. )

    1993-05-01

    A hybrid method incorporating selective polymeric binding and membrane separation was used for the removal of arsenic from contaminated groundwater. Two types of polymers, polyethylenimine (PEI) and poly-diallyl dimethyl ammonium chloride (DADMAC) and an ultrafiltration membrane were used at various concentrations and pH. Bench-scale tests demonstrated a high degree of arsenic removal. The retention of arsenic was also determined in the presence of various background salts such as NaCl, NaNO[sub 3], Na[sub 2]SO[sub 4], Na[sub 2]CO[sub 3]. Retention was found to depend on the concentration and type of anion present in solution. 12 refs., 2 figs., 3 tabs.

  20. Neutron Reflectometry Studies Define Prion Protein N-terminal Peptide Membrane Binding

    PubMed Central

    Le Brun, Anton P.; Haigh, Cathryn L.; Drew, Simon C.; James, Michael; Boland, Martin P.; Collins, Steven J.

    2014-01-01

    The prion protein (PrP), widely recognized to misfold into the causative agent of the transmissible spongiform encephalopathies, has previously been shown to bind to lipid membranes with binding influenced by both membrane composition and pH. Aside from the misfolding events associated with prion pathogenesis, PrP can undergo various posttranslational modifications, including internal cleavage events. Alpha- and beta-cleavage of PrP produces two N-terminal fragments, N1 and N2, respectively, which interact specifically with negatively charged phospholipids at low pH. Our previous work probing N1 and N2 interactions with supported bilayers raised the possibility that the peptides could insert deeply with minimal disruption. In the current study we aimed to refine the binding parameters of these peptides with lipid bilayers. To this end, we used neutron reflectometry to define the structural details of this interaction in combination with quartz crystal microbalance interrogation. Neutron reflectometry confirmed that peptides equivalent to N1 and N2 insert into the interstitial space between the phospholipid headgroups but do not penetrate into the acyl tail region. In accord with our previous studies, interaction was stronger for the N1 fragment than for the N2, with more peptide bound per lipid. Neutron reflectometry analysis also detected lengthening of the lipid acyl tails, with a concurrent decrease in lipid area. This was most evident for the N1 peptide and suggests an induction of increased lipid order in the absence of phase transition. These observations stand in clear contrast to the findings of analogous studies of Ab and α-synuclein and thereby support the possibility of a functional role for such N-terminal fragment-membrane interactions. PMID:25418300

  1. Solubilization and characterization of high-affinity [3H]serotonin binding sites from bovine cortical membranes.

    PubMed Central

    VandenBerg, S R; Allgren, R L; Todd, R D; Ciaranello, R D

    1983-01-01

    High-affinity [3H]serotonin binding activity has been solubilized from bovine cerebral cortical membranes by using Triton X-100, Tween-80, and octyl-beta-D-glucopyranoside. This mixture of detergents solubilizes the high-affinity [3H]serotonin binding activity present in crude membrane preparations with retention of 75-90% specific binding. The detergent mixture was chosen because it can easily be removed from the solubilized fraction by dialysis and polystyrene bead adsorption, thus permitting further purification and isolation of the binding sites. Saturation analysis reveals multiple components of high-affinity [3H]serotonin binding. In crude bovine cortical membranes, at least two binding components are present. A higher-affinity binding component, as defined from curvilinear Scatchard plots, has a Kd for [3H]serotonin of 1-3 nM, whereas a lower-affinity component has a Kd of 10-20 nM. In the solubilized preparation, only a single class of binding sites is apparent, with a Kd of 50-100 nM. Removal of detergents by dialysis and polystyrene bead adsorption results in restoration of the curvilinear Scatchard plot with apparent Kds similar to those observed in crude membrane preparations and with increased Bmax values for each component. [3H]Serotonin binding activity in the solubilized preparation is stable to Sephacryl S-300 column chromatography and to glycerol gradient sedimentation. Saturation analysis of the peak binding fractions from both these procedures once again yields curvilinear Scatchard plots, indicating that the multiple high-affinity binding components are preserved and migrate together. The molecular weight, Stokes radius, and frictional coefficient of the binding site(s) have been calculated. After detergent removal the solubilized material shows many of the characteristics usually attributed to S1 receptors, such as high affinity for [3H]serotonin and its analogs and low affinity for serotonin antagonists. PMID:6574495

  2. Sendai virus-erythrocyte membrane interaction: quantitative and kinetic analysis of viral binding, dissociation, and fusion.

    PubMed

    Hoekstra, D; Klappe, K

    1986-04-01

    A kinetic and quantitative analysis of the binding and fusion of Sendai virus with erythrocyte membranes was performed by using a membrane fusion assay based on the relief of fluorescence self-quenching. At 37 degrees C, the process of virus association displayed a half time of 2.5 min; at 4 degrees C, the half time was 3.0 min. The fraction of the viral dose which became cell associated was independent of the incubation temperature and increased with increasing target membrane concentration. On the average, one erythrocyte ghost can accommodate ca. 1,200 Sendai virus particles. The stability of viral attachment was sensitive to a shift in temperature: a fraction of the virions (ca. 30%), attached at 4 degrees C, rapidly (half time, ca. 2.5 min) eluted from the cell surface at 37 degrees C, irrespective of the presence of free virus in the medium. The elution can be attributed to a spontaneous, temperature-induced release, rather than to viral neuraminidase activity. Competition experiments with nonlabeled virus revealed that viruses destined to fuse do not exchange with free particles in the medium but rather bind in a rapid and irreversible manner. The fusion rate of Sendai virus was affected by the density of the virus particles on the cell surface and became restrained when more than 170 virus particles were attached per ghost. In principle, all virus particles added displayed fusion activity. However, at high virus-to-ghost ratios, only a fraction actually fused, indicating that a limited number of fusion sites exist on the erythrocyte membrane. We estimate that ca. 180 virus particles maximally can fuse with one erythrocyte ghost. PMID:3005662

  3. Delphinium alkaloids as inhibitors of alpha-bungarotoxin binding to rat and insect neural membranes.

    PubMed

    Kukel, C F; Jennings, K R

    1994-01-01

    A series of C19-diterpenoid alkaloids purified from Delphinium were evaluated as inhibitors of alpha-bungarotoxin binding to rat and house fly neural membranes. In comparing these diterpenoid analogs, a wide range of inhibition potencies (IC50) were observed, with calculated IC50 values ranging six orders of magnitude. The most potent inhibitory alkaloids in this series possessed the succinimide aromatic ester moiety in the C18 position. Glaudelsine had an IC50 value of 42 pM at the insect nicotinic acetylcholine receptor. PMID:8012891

  4. Functional binding of a vertebrate hormone, L-3,5,3'-triiodothyronine (T3), on insect follicle cell membranes.

    PubMed

    Kim, Y; Davari, E D; Sevala, V; Davey, K G

    1999-10-01

    A vertebrate hormone, L-3,5,3'-triiodothyronine (T3), induces volume reduction in the follicle cells of Locusta migratoria and Rhodnius prolixus. The effect of T3 on locust follicle cells is inhibited by ouabain and by antibodies raised against a membrane binding protein for juvenile hormone (JH). [125I]-T3 binds to membrane preparations of vitellogenic follicles in a specific and saturable fashion, with a KD in the low nanomolar range. T3 and JH III exhibited equivalent abilities to compete for the T3 binding site. These findings strongly suggest that T3 and JH act via the same receptor in follicle cells. PMID:10528413

  5. Organization into Higher Ordered Ring Structures Counteracts Membrane Binding of IM30, a Protein Associated with Inner Membranes in Chloroplasts and Cyanobacteria.

    PubMed

    Heidrich, Jennifer; Wulf, Verena; Hennig, Raoul; Saur, Michael; Markl, Jürgen; Sönnichsen, Carsten; Schneider, Dirk

    2016-07-15

    The IM30 (inner membrane-associated protein of 30 kDa), also known as the Vipp1 (vesicle-inducing protein in plastids 1), has a crucial role in thylakoid membrane biogenesis and maintenance. Recent results suggest that the protein binds peripherally to membranes containing negatively charged lipids. However, although IM30 monomers interact and assemble into large oligomeric ring complexes with different numbers of monomers, it is still an open question whether ring formation is crucial for membrane interaction. Here we show that binding of IM30 rings to negatively charged phosphatidylglycerol membrane surfaces results in a higher ordered membrane state, both in the head group and in the inner core region of the lipid bilayer. Furthermore, by using gold nanorods covered with phosphatidylglycerol layers and single particle spectroscopy, we show that not only IM30 rings but also lower oligomeric IM30 structures interact with membranes, although with higher affinity. Thus, ring formation is not crucial for, and even counteracts, membrane interaction of IM30. PMID:27226585

  6. Nonspecific Binding Domains in Lipid Membranes Induced by Phospholipase A2.

    PubMed

    Hong, Chia Yee; Han, Chung-Ta; Chao, Ling

    2016-07-12

    Phospholipase A2 (PLA2) is a peripheral membrane protein that can hydrolyze phospholipids to produce lysolipids and fatty acids. It has been found to play crucial roles in various cellular processes and is thought as a potential candidate for triggering drug release from liposomes for medical treatment. Here, we directly observed that PLA2 hydrolysis reaction can induce the formation of PLA2-binding domains at lipid bilayer interface and found that the formation was significantly influenced by the fluidity of the lipid bilayer. We prepared supported lipid bilayers (SLBs) with various molar ratios of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) to adjust the reactivity and fluidity of the lipid bilayers. A significant amount of the PLA2-induced domains was observed in mixtures of DPPC and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) but not in either pure DPPC or pure DOPC bilayer, which might be the reason that previous studies rarely observed these domains in lipid bilayer systems. The fluorescently labeled PLA2 experiment showed that newly formed domains acted as binding templates for PLA2. The AFM result showed that the induced domain has stepwise plateau structure, suggesting that PLA2 hydrolysis products may align as bilayers and accumulate layer by layer on the support, and the hydrophobic acyl chains at the side of the layer structure may be exposed to the outside aqueous environment. The introduced hydrophobic region could have hydrophobic interactions with proteins and therefore can attract the binding of not only PLA2 but also other types of proteins such as proteoglycans and streptavidin. The results suggest that the formation of PLA2-induced domains may convert part of a zwitterionic nonsticky lipid membrane to a site where biomolecules can nonspecifically bind. PMID:27218880

  7. Label-free probe of HIV-1 TAT peptide binding to mimetic membranes.

    PubMed

    Rao, Yi; Kwok, Sheldon J J; Lombardi, Julien; Turro, Nicholas J; Eisenthal, Kenneth B

    2014-09-01

    The transacting activator of transduction (TAT) protein plays a key role in the progression of AIDS. Studies have shown that a +8 charged sequence of amino acids in the protein, called the TAT peptide, enables the TAT protein to penetrate cell membranes. To probe mechanisms of binding and translocation of the TAT peptide into the cell, investigators have used phospholipid liposomes as cell membrane mimics. We have used the method of surface potential sensitive second harmonic generation (SHG), which is a label-free and interface-selective method, to study the binding of TAT to anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-1'-rac-glycerol (POPG) and neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes. It is the SHG sensitivity to the electrostatic field generated by a charged interface that enabled us to obtain the interfacial electrostatic potential. SHG together with the Poisson-Boltzmann equation yielded the dependence of the surface potential on the density of adsorbed TAT. We obtained the dissociation constants Kd for TAT binding to POPC and POPG liposomes and the maximum number of TATs that can bind to a given liposome surface. For POPC Kd was found to be 7.5 ± 2 μM, and for POPG Kd was 29.0 ± 4.0 μM. As TAT was added to the liposome solution the POPC surface potential changed from 0 mV to +37 mV, and for POPG it changed from -57 mV to -37 mV. A numerical calculation of Kd, which included all terms obtained from application of the Poisson-Boltzmann equation to the TAT liposome SHG data, was shown to be in good agreement with an approximated solution. PMID:25136100

  8. Characterization of membrane-bound small GTP-binding proteins from Nicotiana tabacum.

    PubMed Central

    Haizel, T; Merkle, T; Turck, F; Nagy, F

    1995-01-01

    We have cloned nine cDNAs encoding small GTP-binding proteins from leaf cDNA libraries of tobacco (Nicotiana tabacum). These cDNAs encode distinct proteins (22-25 kD) that display different levels of identity with members of the mammalian Rab family: Nt-Rab6 with Rab6 (83%), Nt-Rab7a-c with Rab7 (63-70%), and Nt-Rab11a-e with Rab11 (53-69%). Functionally important regions of these proteins, including the "effector binding" domain, the C-terminal Cys residues for membrane attachment, and the four regions involved in GTP-binding and hydrolysis, are highly conserved. Northern and western blot analyses show that these genes are expressed, although at slightly different levels, in all plant tissues examined. We demonstrate that the plant Rab5, Rab6, and Rab11 proteins, similar to their mammalian and yeast counterparts, are tightly bound to membranes and that they exhibit different solubilization characteristics. Furthermore, we show that the yeast GTPase-activating protein Gyp6, shown to be specifically required to control the GTP hydrolysis of the yeast Ypt6 protein, could interact with tobacco GTP-binding proteins. It increases in vitro the GTP hydrolysis rate of the wild-type Nt-Rab7 protein. In addition, it also increases, at different levels, the GTP hydrolysis rates of a Nt-Rab7m protein with a Rab6 effector domain and of two other chimaeric Nt-Rab6/Nt-Rab7 proteins. However, it does not interact with the wild-type Nt-Rab6 protein, which is most similar to the yeast Ypt6 protein. PMID:7784525

  9. Label-free probe of HIV-1 TAT peptide binding to mimetic membranes

    PubMed Central

    Rao, Yi; Kwok, Sheldon J. J.; Lombardi, Julien; Turro, Nicholas J.; Eisenthal, Kenneth B.

    2014-01-01

    The transacting activator of transduction (TAT) protein plays a key role in the progression of AIDS. Studies have shown that a +8 charged sequence of amino acids in the protein, called the TAT peptide, enables the TAT protein to penetrate cell membranes. To probe mechanisms of binding and translocation of the TAT peptide into the cell, investigators have used phospholipid liposomes as cell membrane mimics. We have used the method of surface potential sensitive second harmonic generation (SHG), which is a label-free and interface-selective method, to study the binding of TAT to anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-1′-rac-glycerol (POPG) and neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes. It is the SHG sensitivity to the electrostatic field generated by a charged interface that enabled us to obtain the interfacial electrostatic potential. SHG together with the Poisson–Boltzmann equation yielded the dependence of the surface potential on the density of adsorbed TAT. We obtained the dissociation constants Kd for TAT binding to POPC and POPG liposomes and the maximum number of TATs that can bind to a given liposome surface. For POPC Kd was found to be 7.5 ± 2 μM, and for POPG Kd was 29.0 ± 4.0 μM. As TAT was added to the liposome solution the POPC surface potential changed from 0 mV to +37 mV, and for POPG it changed from −57 mV to −37 mV. A numerical calculation of Kd, which included all terms obtained from application of the Poisson–Boltzmann equation to the TAT liposome SHG data, was shown to be in good agreement with an approximated solution. PMID:25136100

  10. Binding of E. coli heat-stable enterotoxin to rat intestinal brush borders and to basolateral membranes

    SciTech Connect

    Guarino, A.; Cohen, M.B.; Overmann, G.; Thompson, M.R.; Giannella, R.A.

    1987-09-01

    We studied the binding of E. coli heat-stable enterotoxin (STa) to rat brush borders (BB) and to basolateral membranes (BLM) using a biologically active monoiodinated radioligand (( /sup 125/I)STa) and highly enriched BB and BLM preparations free of other significant organelle contamination. Binding of (/sup 125/I)STa to BB was specific; time-, temperature-, and pH-dependent; saturable; and partially reversible. Nonlabeled toxin competitively inhibited the binding of radioligand to BB in a dose-related manner. Scatchard analysis revealed a single class of receptors with an apparent affinity constant of 8.7 +/- 1.5 X 10(8) l/mol. Binding was not affected by amino acids, sugars, and lectins. Proteolytic enzymes significantly decreased binding, although several did so by modifying the radioligand. Trypsin inhibited binding without modifying the radioligand thus supporting the proteinaceous nature of the receptor. Since the enrichment in binding activity in the BB over the homogenate was significantly lower than the enrichment in sucrase activity, we concluded that binding activity is probably associated with other membranous domains, but direct examination revealed no binding activity on basolateral membranes.

  11. Structural insights into the methyl donor recognition model of a novel membrane-binding protein UbiG

    PubMed Central

    Zhu, Yuwei; Jiang, Xuguang; Wang, Chongyuan; Liu, Yang; Fan, Xiaojiao; Zhang, Linjuan; Niu, Liwen; Teng, Maikun; Li, Xu

    2016-01-01

    UbiG is a SAM-dependent O-methyltransferase, catalyzing two O-methyl transfer steps for ubiquinone biosynthesis in Escherichia coli. UbiG possesses a unique sequence insertion between β4 and α10, which is used for membrane lipid interaction. Interestingly, this sequence insertion also covers the methyl donor binding pocket. Thus, the relationship between membrane binding and entrance of the methyl donor of UbiG during the O-methyl transfer process is a question that deserves further exploration. In this study, we reveal that the membrane-binding region of UbiG gates the entrance of methyl donor. When bound with liposome, UbiG displays an enhanced binding ability toward the methyl donor product S-adenosylhomocysteine. We further employ protein engineering strategies to design UbiG mutants by truncating the membrane interacting region or making it more flexible. The ITC results show that the binding affinity of these mutants to SAH increases significantly compared with that of the wild-type UbiG. Moreover, we determine the structure of UbiG∆165–187 in complex with SAH. Collectively, our results provide a new angle to cognize the relationship between membrane binding and entrance of the methyl donor of UbiG, which is of benefit for better understanding the O-methyl transfer process for ubiquinone biosynthesis. PMID:26975567

  12. Circulating IgM Requires Plasma Membrane Disruption to Bind Apoptotic and Non-Apoptotic Nucleated Cells and Erythrocytes

    PubMed Central

    Hesketh, Emily E.; Dransfield, Ian; Kluth, David C.; Hughes, Jeremy

    2015-01-01

    Autoimmunity is associated with defective phagocytic clearance of apoptotic cells. IgM deficient mice exhibit an autoimmune phenotype consistent with a role for circulating IgM antibodies in apoptotic cell clearance. We have extensively characterised IgM binding to non-apoptotic and apoptotic mouse thymocytes and human Jurkat cells using flow cytometry, confocal imaging and electron microscopy. We demonstrate strong specific IgM binding to a subset of Annexin-V (AnnV)+PI (Propidium Iodide)+ apoptotic cells with disrupted cell membranes. Electron microscopy studies indicated that IgM+AnnV+PI+ apoptotic cells exhibited morphologically advanced apoptosis with marked plasma membrane disruption compared to IgM-AnnV+PI+ apoptotic cells, suggesting that access to intracellular epitopes is required for IgM to bind. Strong and comparable binding of IgM to permeabilised non-apoptotic and apoptotic cells suggests that IgM bound epitopes are 'apoptosis independent' such that IgM may bind any cell with profound disruption of cell plasma membrane integrity. In addition, permeabilised erythrocytes exhibited significant IgM binding thus supporting the importance of cell membrane epitopes. These data suggest that IgM may recognize and tag damaged nucleated cells or erythrocytes that exhibit significant cell membrane disruption. The role of IgM in vivo in conditions characterized by severe cell damage such as ischemic injury, sepsis and thrombotic microangiopathies merits further exploration. PMID:26121639

  13. The effect of diffusion on the binding of membrane-bound receptors to coated pits.

    PubMed Central

    Keizer, J; Ramirez, J; Peacock-López, E

    1985-01-01

    We have formulated a kinetic model for the primary steps that occur at the cell membrane during receptor-mediated endocytosis. This model includes the diffusion of receptor molecules, the binding of receptors to coated pits, the loss of coated pits by invagination, and random reinsertion of receptors and coated pits. Using the mechanistic statistical theory of nonequilibrium thermodynamics, we employ this mechanism to calculate the two-dimensional radial distribution of receptors around coated pits at steady state. From this we obtain an equation that describes the effect of receptor diffusion on the rate of binding to coated pits. Our equation does not assume that ligand binding is instantaneous and can be used to assess the effect of diffusion on the binding rate. Using experimental data for low density lipoprotein receptors on fibroblast cells, we conclude that the effect of diffusion on the binding of these receptors to coated pits is no more than 84% diffusion controlled. This corresponds to a dissociation rate constant for receptors on coated pits (k-) that is much less than the rate constant for invagination of the pits (lambda = 3.3 X 10(-3)/s) and a correlation length for the radial distribution function of six times the radius of a coated pit. Although the existing experimental data are compatible with any value of k-, we obtain a lower bound for the value of the binding constant (k+) of 2.3 X 10(-2)(micron)2/s. Comparison of the predicted radial distributions with experiment should provide a clear indication of the effect of diffusion on k+. PMID:2858230

  14. The Relationship between Glycan Binding and Direct Membrane Interactions in Vibrio cholerae Cytolysin, a Channel-forming Toxin.

    PubMed

    De, Swastik; Bubnys, Adele; Alonzo, Francis; Hyun, Jinsol; Lary, Jeffrey W; Cole, James L; Torres, Victor J; Olson, Rich

    2015-11-20

    Bacterial pore-forming toxins (PFTs) are structurally diverse pathogen-secreted proteins that form cell-damaging channels in the membranes of host cells. Most PFTs are released as water-soluble monomers that first oligomerize on the membrane before inserting a transmembrane channel. To modulate specificity and increase potency, many PFTs recognize specific cell surface receptors that increase the local toxin concentration on cell membranes, thereby facilitating channel formation. Vibrio cholerae cytolysin (VCC) is a toxin secreted by the human pathogen responsible for pandemic cholera disease and acts as a defensive agent against the host immune system. Although it has been shown that VCC utilizes specific glycan receptors on the cell surface, additional direct contacts with the membrane must also play a role in toxin binding. To better understand the nature of these interactions, we conducted a systematic investigation of the membrane-binding surface of VCC to identify additional membrane interactions important in cell targeting. Through cell-based assays on several human-derived cell lines, we show that VCC is unlikely to utilize high affinity protein receptors as do structurally similar toxins from Staphylococcus aureus. Next, we identified a number of specific amino acid residues that greatly diminish the VCC potency against cells and investigated the interplay between glycan binding and these direct lipid contacts. Finally, we used model membranes to parse the importance of these key residues in lipid and cholesterol binding. Our study provides a complete functional map of the VCC membrane-binding surface and insights into the integration of sugar, lipid, and cholesterol binding interactions. PMID:26416894

  15. Detection and quantification of lipid membrane binding on silica micro-tube resonator sensor

    NASA Astrophysics Data System (ADS)

    Ling, Tao; Majd, Sheereen; Mayer, Michael; Guo, L. Jay

    2008-02-01

    Silica micro-tube resonator is a very attractive biosensor platform for label-free detection of bio-molecules by combining high sensitivity and simple fluidic handling. We report the study of lipid membrane binding on micro-tube sensor that is probed by a prism coupling technique. Prism coupling to the micro-tube resonance modes allows thick micro-tube to be used and the selectivity of high-sensitivity modes. We were able to identify and probe a special resonance mode that has very high Electrical field at the boundary of the fluid and the inner tube wall. Unlike typical WGM modes, such resonance modes also have very high Electrical field extending into the lower index fluid region, thereby providing exceptionally high sensitivity to the fluid's refractive index change. We used this type of resonance mode to detect the formation of single bilayer lipid membrane on the inner tube wall. With 4-5nm POPC lipid membrane with refractive index around 1.46 absorbed on the inner wall, we can observe the resonance peak shift around 44pm. Mie scattering simulation of the resonance peak shift due to the bio-film absorbed onto the inner wall agrees very well with the experiment results. We also observed resonance peak shift with the membrane protein Annexin V bonding to the lipid membrane. With the present Q factor 6×10 4 at 1.55μm wavelength, we estimate that our devices can detect the presence of 0.1nm thick absorbed film on the inner wall of the tube. The device's sensitivity can be greatly enhanced by switching the working wavelength from infrared wavelength to visible wavelength where water absorption is minimized.

  16. Membrane binding mode of intrinsically disordered cytoplasmic domains of T cell receptor signaling subunits depends on lipid composition

    SciTech Connect

    Sigalov, Alexander B.; Hendricks, Gregory M.

    2009-11-13

    Intrinsically disordered cytoplasmic domains of T cell receptor (TCR) signaling subunits including {zeta}{sub cyt} and CD3{epsilon}{sub cyt} all contain one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor triggering. Membrane binding-induced helical folding of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} ITAMs is thought to control TCR activation. However, the question whether or not lipid binding of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} is necessarily accompanied by a folding transition of ITAMs remains open. In this study, we investigate whether the membrane binding mechanisms of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} depend on the membrane model used. Circular dichroic and fluorescence data indicate that binding of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} to detergent micelles and unstable vesicles is accompanied by a disorder-to-order transition, whereas upon binding to stable vesicles these proteins remain unfolded. Using electron microscopy and dynamic light scattering, we show that upon protein binding, unstable vesicles fuse and rupture. In contrast, stable vesicles remain intact under these conditions. This suggests different membrane binding modes for {zeta}{sub cyt} and CD3{epsilon}{sub cyt} depending on the bilayer stability: (1) coupled binding and folding, and (2) binding without folding. These findings explain the long-standing puzzle in the literature and highlight the importance of the choice of an appropriate membrane model for protein-lipid interactions studies.

  17. Photosensitizer binding to lipid bilayers as a precondition for the photoinactivation of membrane channels.

    PubMed Central

    Rokitskaya, T I; Block, M; Antonenko, Y N; Kotova, E A; Pohl, P

    2000-01-01

    The photodynamic activity of sulfonated aluminum phthalocyanines (AlPcS(n), 1 membrane lipids. Adsorbing to the surface of large unilamellar vesicles (LUVs), aluminum phthalocyanine disulfonate induced the highest changes in their electrophoretic mobility. AlPcS(2) was also most efficient in mediating photoinactivation of gramicidin channels, as revealed by measurements of the electric current across planar lipid bilayers. The increase in the degree of sulfonation of phthalocyanine progressively reduced its affinity for the lipid bilayer as well as its potency of sensitizing gramicidin channel photoinactivation. The portion of photoinactivated gramicidin channels, alpha, increased with rising photosensitizer concentration up to some optimum. The concentration at which alpha was at half-maximum amounted to 80 nM, 30 nM, 200 nM, and 2 microM for AlPcS(1), AlPcS(2), AlPcS(3), and AlPcS(4), respectively. At high concentrations alpha was found to decrease, which was attributed to quenching of reactive oxygen species and self-quenching of the photosensitizer triplet state by its ground state. Fluoride anions were observed to inhibit both AlPcS(n) (2 binding to LUVs and sensitized photoinactivation of gramicidin channels. It is concluded that photosensitizer binding to membrane lipids is a prerequisite for the photodynamic inactivation of gramicidin channels. PMID:10777753

  18. Binding of ( sup 125 I)iodipine to parathyroid cell membranes: Evidence of a dihydropyridine-sensitive calcium channel

    SciTech Connect

    Jones, J.I.; Fitzpatrick, L.A. )

    1990-04-01

    The parathyroid cell is unusual, in that an increase in extracellular calcium concentrations inhibits PTH release. Calcium channels are glycoproteins that span cell membranes and allow entry of extracellular calcium into cells. We have demonstrated that the calcium channel agonist (+)202-791, which opens calcium channels, inhibits PTH release and that the antagonist (-)202-791, which closes calcium channels, stimulates PTH release. To identify the calcium channels responsible for these effects, we used a radioligand that specifically binds to calcium channels. Bovine parathyroid cell membranes were prepared and incubated under reduced lighting with (125I) iodipine (SA, 2000 Ci/mmol), which recognizes 1,4-dihydropyridine-sensitive calcium channels. Bound ligand was separated from free ligand by rapid filtration through Whatman GF/B filters. Nonspecific binding was measured by the inclusion of nifedipine at 10 microM. Specific binding represented approximately 40% of the total binding. The optimal temperature for (125I) iodipine binding was 4 C, and binding reached equilibrium by 30 min. The equilibrium dissociation constant (Kd) was approximately 550 pM, and the maximum number of binding sites was 780 fmol/mg protein. Both the calcium channel agonist (+)202-791 and antagonist (-)202-791 competitively inhibited (125I) iodipine binding, with 50% inhibition concentrations of 20 and 300 nM, respectively. These data indicate the presence of dihydropyridine-sensitive calcium channels on parathyroid cell membranes.

  19. Impact of Reducing Complement Inhibitor Binding on the Immunogenicity of Native Neisseria meningitidis Outer Membrane Vesicles

    PubMed Central

    Daniels-Treffandier, Helene; de Nie, Karlijn; Marsay, Leanne; Dold, Christina; Sadarangani, Manish; Reyes-Sandoval, Arturo; Langford, Paul R.; Wyllie, David; Hill, Fergal; Pollard, Andrew J.; Rollier, Christine S.

    2016-01-01

    Neisseria meningitidis recruits host human complement inhibitors to its surface to down-regulate complement activation and enhance survival in blood. We have investigated whether such complement inhibitor binding occurs after vaccination with native outer membrane vesicles (nOMVs), and limits immunogenicity of such vaccines. To this end, nOMVs reactogenic lipopolysaccharide was detoxified by deletion of the lpxl1 gene (nOMVlpxl1). nOMVs unable to bind human complement factor H (hfH) were generated by additional deletions of the genes encoding factor H binding protein (fHbp) and neisserial surface protein A (NspA) (nOMVdis). Antibody responses elicited in mice with nOMVdis were compared to those elicited with nOMVlpxl1 in the presence of hfH. Results demonstrate that the administration of human fH to mice immunized with fHbp containing OMVlpxl1 decreased immunogenicity against fHbp (but not against the OMV as a whole). The majority of the OMV-induced bactericidal immune response (OMVlpxl1 or OMVdis) was versus PorA. Despite a considerable reduction of hfH binding to nOMVdis, and the absence of the vaccine antigen fHbp, immunogenicity in mice was not different from nOMVlpxl1, in the absence or presence of hfH (serum bactericidal titers of 1:64 vs 1:128 after one dose in the nOMVdis and nOMVlpxl1–immunized groups respectively). Therefore, partial inhibition of fH binding did not enhance immunity in this model. PMID:26871712

  20. Impact of Reducing Complement Inhibitor Binding on the Immunogenicity of Native Neisseria meningitidis Outer Membrane Vesicles.

    PubMed

    Daniels-Treffandier, Helene; de Nie, Karlijn; Marsay, Leanne; Dold, Christina; Sadarangani, Manish; Reyes-Sandoval, Arturo; Langford, Paul R; Wyllie, David; Hill, Fergal; Pollard, Andrew J; Rollier, Christine S

    2016-01-01

    Neisseria meningitidis recruits host human complement inhibitors to its surface to down-regulate complement activation and enhance survival in blood. We have investigated whether such complement inhibitor binding occurs after vaccination with native outer membrane vesicles (nOMVs), and limits immunogenicity of such vaccines. To this end, nOMVs reactogenic lipopolysaccharide was detoxified by deletion of the lpxl1 gene (nOMVlpxl1). nOMVs unable to bind human complement factor H (hfH) were generated by additional deletions of the genes encoding factor H binding protein (fHbp) and neisserial surface protein A (NspA) (nOMVdis). Antibody responses elicited in mice with nOMVdis were compared to those elicited with nOMVlpxl1 in the presence of hfH. Results demonstrate that the administration of human fH to mice immunized with fHbp containing OMVlpxl1 decreased immunogenicity against fHbp (but not against the OMV as a whole). The majority of the OMV-induced bactericidal immune response (OMVlpxl1 or OMVdis) was versus PorA. Despite a considerable reduction of hfH binding to nOMVdis, and the absence of the vaccine antigen fHbp, immunogenicity in mice was not different from nOMVlpxl1, in the absence or presence of hfH (serum bactericidal titers of 1:64 vs 1:128 after one dose in the nOMVdis and nOMVlpxl1-immunized groups respectively). Therefore, partial inhibition of fH binding did not enhance immunity in this model. PMID:26871712

  1. Using Micropatterned Lipid Bilayer Arrays to Measure the Effect of Membrane Composition on Merocyanine 540 Binding

    PubMed Central

    Smith, Kathryn A.; Conboy, John C.

    2011-01-01

    The lipophilic dye merocyanine 540 (MC540) was used to model small molecule-membrane interactions using micropatterned lipid bilayer arrays (MLBAs) prepared using a 3D Continuous Flow Microspotter (CFM). Fluorescence microscopy was used to monitor MC540 binding to fifteen different bilayer compositions simultaneously. MC540 fluorescence was two times greater for bilayers composed of liquid-crystalline (l.c.) phase lipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) compared to bilayers in the gel phase (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)). The effect cholesterol (CHO) had on MC540 binding to the membrane was found to be dependent on the lipid component; cholesterol decreased MC540 bindingin DMPC, DPPC and DSPC bilayers while having little to no effect on the remaining l.c. phase lipids. MC540 fluorescence was also lowered when 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS) was incorporated into DOPC bilayers. The increase in the surface charge density appears to decrease the occurrence of highly fluorescent monomers and increase the formation of weakly fluorescent dimers via electrostatic repulsion. This paper demonstrates that MLBAs are a useful tool for preparing high density reproducible bilayer arrays to study small molecule-membrane interactions in a high-throughput manner. PMID:21376014

  2. BB0172, a Borrelia burgdorferi Outer Membrane Protein That Binds Integrin α3β1

    PubMed Central

    Wood, Elaine; Tamborero, Silvia; Mingarro, Ismael

    2013-01-01

    Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi infection. Upon infection, some B. burgdorferi genes are upregulated, including members of the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) protein family, which facilitate B. burgdorferi adherence to extracellular matrix components of the host. Comparative genome analysis has revealed a new family of B. burgdorferi proteins containing the von Willebrand factor A (vWFA) domain. In the present study, we characterized the expression and membrane association of the vWFA domain-containing protein BB0172 by using in vitro transcription/translation systems in the presence of microsomal membranes and with detergent phase separation assays. Our results showed evidence of BB0172 localization in the outer membrane, the orientation of the vWFA domain to the extracellular environment, and its function as a metal ion-dependent integrin-binding protein. This is the first report of a borrelial adhesin with a metal ion-dependent adhesion site (MIDAS) motif that is similar to those observed in eukaryotic integrins and has a similar function. PMID:23687274

  3. A large scale membrane-binding protein conformational change that initiates at small length scales

    NASA Astrophysics Data System (ADS)

    Grandpre, Trevor; Andorf, Matthew; Chakravarthy, Srinivas; Lamb, Robert; Poor, Taylor; Landahl, Eric

    2013-03-01

    The fusion (F) protein of parainfluenza virus 5 (PIV5) is a membrane-bound, homotrimeric glycoprotein located on the surface of PIV5 viral envelopes. Upon being triggered by the receptor-binding protein (HN), F undergoes a greater than 100Å ATP-independent refolding event. This refolding event results in the insertion of a hydrophobic fusion peptide into the membrane of the target cell, followed by the desolvation and subsequent fusion event as the two membranes are brought together. Isothermal calorimetry and hydrophobic dye incorporation experiments indicate that the soluble construct of the F protein undergoes a conformational rearrangement event at around 55 deg C. We present the results of an initial Time-Resolved Small-Angle X-Ray Scattering (TR-SAXS) study of this large scale, entropically driven conformational change using a temperature jump. Although we the measured radius of gyration of this protein changes on a 110 second timescale, we find that the x-ray scattering intensity at higher angles (corresponding to smaller length scales in the protein) changes nearly an order of magnitude faster. We believe this may be a signature of entropically-driven conformational change. To whom correspondence should be addressed

  4. BB0172, a Borrelia burgdorferi outer membrane protein that binds integrin α3β1.

    PubMed

    Wood, Elaine; Tamborero, Silvia; Mingarro, Ismael; Esteve-Gassent, Maria D

    2013-08-01

    Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi infection. Upon infection, some B. burgdorferi genes are upregulated, including members of the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) protein family, which facilitate B. burgdorferi adherence to extracellular matrix components of the host. Comparative genome analysis has revealed a new family of B. burgdorferi proteins containing the von Willebrand factor A (vWFA) domain. In the present study, we characterized the expression and membrane association of the vWFA domain-containing protein BB0172 by using in vitro transcription/translation systems in the presence of microsomal membranes and with detergent phase separation assays. Our results showed evidence of BB0172 localization in the outer membrane, the orientation of the vWFA domain to the extracellular environment, and its function as a metal ion-dependent integrin-binding protein. This is the first report of a borrelial adhesin with a metal ion-dependent adhesion site (MIDAS) motif that is similar to those observed in eukaryotic integrins and has a similar function. PMID:23687274

  5. Autoradiographic localization of a non-reducible somatostatin analog (/sup 125/I-CGP 23996) binding sites in the rat brain: comparison with membrane binding

    SciTech Connect

    Epelbaum, J.; Dussaillant, M.; Enjalbert, A.; Kordon, C.; Rostene, W.

    1985-07-01

    The regional distribution of somatostatin binding sites in the rat brain was determined by quantitative autoradiography, using /sup 125/I-CGP 23996, a non-reducible somatostatin analog. In preliminary experiments, kinetic properties of /sup 125/I-CGP 23996 binding to rat brain membranes and slide mounted frozen brain sections were compared and found similar. In addition, distribution of /sup 125/I-CGP 23996 and /sup 125/I-N-Tyr-SRIF14 binding sites on membrane prepared from 10 different rat brain structures were closely correlated (r = 0.91, 2 p less than 0.01), indicating that the non-reducible analog recognizes the same binding site as the Tyr-extended native peptide. Highest levels of /sup 125/I-CGP 23996 binding sites were found in anterior temporal, frontal and cingular cortex as well as hippocampus. Moderate levels were found in the remaining part of the limbic system including amygdala, olfactory tubercles and bed nucleus of the stria terminalis. In the brain stem, nuclei involved in the auditory system such as the ventral cochlear nucleus and the superior olive nucleus, contained high levels of /sup 125/I-CGP 23996 binding sites. The distribution of /sup 125/I-CGP 23996 binding sites roughly correlated with that of the endogenous peptide in most structures, except in the mediobasal hypothalamus.

  6. Novel structures in secreting the androgenic gland hormone.

    PubMed

    Negishi, S; Hasegawa, Y; Nakajima, Y

    2001-12-01

    The secretory granules in the androgenic gland of the terrestrial isopod Armadillidium vulgare, which have been indistinct for long time because of vulnerable structures, were revealed by using the rapid-freezing and freeze-substitution method. The fine structure of the androgenic gland is conspicuous by the distribution of numerous particular organelles in the cytoplasm consisting of the endoplasmic reticulum and the Golgi complex, and by having a number of highly organized structures developed between the androgenic gland cells. The structures connect to the intercellular space, which is seen as intercellular canaliculi for exporting the androgenic gland hormone. The plasma membranes near the particular structure of the intercellular canaliculi in the androgenic gland are often specialized to form cellular junctions. The secretory granules including the electron-dense materials, which are supposed to be peptides of androgenic gland hormone, are distributed beside the particular structure of the intercellular canaliculi. Some of the granules are seen to fuse with the plasma membranes. This observation suggests that, in the Armadillidium vulgare, the secretory granules containing androgenic gland hormone are transferred to the extracellular space through the intercellular canaliculi particularly developed for exporting the peptide hormone. This is the first evidence to show the secretory mechanism of the androgenic gland hormone in the Isopoda. PMID:11911080

  7. Characterization of the vasoactive intestinal peptide receptor in rat submandibular gland: radioligand binding assay in membrane preparations

    SciTech Connect

    Turner, J.T.; Bylund, D.B.

    1987-09-01

    The vasoactive intestinal peptide (VIP) receptor in membranes from rat submandibular gland was studied using radioligand binding assays with /sup 125/I-VIP and various unlabeled competing ligands. In addition to the necessity of working within the parameters under which all radioligand binding assays should be performed, binding studies with /sup 125/I-VIP, as with other peptide hormones and neurotransmitters, are subject to additional technical difficulties. Specific problems that were addressed included radioligand proteolysis, the identification of an effective protease inhibitor (leupeptin) and the deleterious effects of a commonly used inhibitor (bacitracin); avid radioligand absorption to incubation tubes that was eliminated by precoating of the tubes with a combination of polyethylenimine and an organosilane; and a disproportionate effect of increasing membrane protein concentration on affinity estimates. Under optimized conditions, the affinity (Kd) and density Bmax values for /sup 125/I-VIP obtained from saturation assays (76 pM, 2.0 pmol/mg) were in excellent agreement. Membrane protein (or receptor) levels beyond the linear portion of the receptor concentration curve are often used in radioligand binding assays. Results from /sup 125/I-VIP binding studies at elevated receptor concentrations revealed the predicted marked decrease in receptor affinity. In addition, the rank order potency of unlabeled ligands in inhibition binding assays was changed. The optimization of the assay for measuring VIP receptors in submandibular gland membrane provides a reliable method for studying the role of receptor regulation in stimulus-secretion coupling for this neuropeptide.

  8. Effect of anchor positioning on binding and diffusion of elongated 3D DNA nanostructures on lipid membranes

    NASA Astrophysics Data System (ADS)

    Khmelinskaia, Alena; Franquelim, Henri G.; Petrov, Eugene P.; Schwille, Petra

    2016-05-01

    DNA origami is a state-of-the-art technology that enables the fabrication of nano-objects with defined shapes, to which functional moieties, such as lipophilic anchors, can be attached with a nanometre scale precision. Although binding of DNA origami to lipid membranes has been extensively demonstrated, the specific requirements necessary for membrane attachment are greatly overlooked. Here, we designed a set of amphipathic rectangular-shaped DNA origami structures with varying placement and number of chol-TEG anchors used for membrane attachment. Single- and multiple-cholesteryl-modified origami nanostructures were produced and studied in terms of their membrane localization, density and dynamics. We show that the positioning of at least two chol-TEG moieties near the corners is essential to ensure efficient membrane binding of large DNA nanostructures. Quantitative fluorescence correlation spectroscopy data further confirm that increasing the number of corner-positioned chol-TEG anchors lowers the dynamics of flat DNA origami structures on freestanding membranes. Taken together, our approach provides the first evidence of the importance of the location in addition to the number of hydrophobic moieties when rationally designing minimal DNA nanostructures with controlled membrane binding.

  9. Binding constants of membrane-anchored receptors and ligands: A general theory corroborated by Monte Carlo simulations.

    PubMed

    Xu, Guang-Kui; Hu, Jinglei; Lipowsky, Reinhard; Weikl, Thomas R

    2015-12-28

    Adhesion processes of biological membranes that enclose cells and cellular organelles are essential for immune responses, tissue formation, and signaling. These processes depend sensitively on the binding constant K2D of the membrane-anchored receptor and ligand proteins that mediate adhesion, which is difficult to measure in the "two-dimensional" (2D) membrane environment of the proteins. An important problem therefore is to relate K2D to the binding constant K3D of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in three dimensions (3D). In this article, we present a general theory for the binding constants K2D and K3D of rather stiff proteins whose main degrees of freedom are translation and rotation, along membranes and around anchor points "in 2D," or unconstrained "in 3D." The theory generalizes previous results by describing how K2D depends both on the average separation and thermal nanoscale roughness of the apposing membranes, and on the length and anchoring flexibility of the receptors and ligands. Our theoretical results for the ratio K2D/K3D of the binding constants agree with detailed results from Monte Carlo simulations without any data fitting, which indicates that the theory captures the essential features of the "dimensionality reduction" due to membrane anchoring. In our Monte Carlo simulations, we consider a novel coarse-grained model of biomembrane adhesion in which the membranes are represented as discretized elastic surfaces, and the receptors and ligands as anchored molecules that diffuse continuously along the membranes and rotate at their anchor points. PMID:26723621

  10. Identification of Escherichia coli F4ac-binding proteins in porcine milk fat globule membrane.

    PubMed

    Novakovic, Predrag; Huang, Yanyun Y; Lockerbie, Betty; Shahriar, Farshid; Kelly, John; Gordon, John R; Middleton, Dorothy M; Loewen, Matthew E; Kidney, Beverly A; Simko, Elemir

    2015-04-01

    F4ac-positive enterotoxigenic Escherichia coli (ETEC) must attach to the intestinal mucosa to cause diarrhea in piglets. Prevention of bacterial attachment to the intestinal mucosa is the most effective defense against ETEC-induced diarrhea. Porcine milk fat globule membranes (MFGM) were shown to be able to inhibit attachment of ETEC to the intestinal brush border; however, the specific components of porcine MFGM that inhibited attachment of ETEC to enterocytes were not identified. Accordingly, the purpose of this study was to identify F4ac-binding MFGM proteins by overlay Western blot and affinity chromatography. The proteome of porcine MFGM was characterized and the following F4ac-binding proteins were detected by overlay Western blot and affinity chromatography: lactadherin, butyrophilin, adipophilin, acyl-CoA synthetase 3, and fatty acid-binding protein 3. The biological function of these proteins was not investigated but it is possible that their interaction with F4ac fimbria interferes with bacterial attachment and colonization. PMID:25852227

  11. Identification of Escherichia coli F4ac-binding proteins in porcine milk fat globule membrane

    PubMed Central

    Novakovic, Predrag; Huang, Yanyun Y.; Lockerbie, Betty; Shahriar, Farshid; Kelly, John; Gordon, John R.; Middleton, Dorothy M.; Loewen, Matthew E.; Kidney, Beverly A.; Simko, Elemir

    2015-01-01

    F4ac-positive enterotoxigenic Escherichia coli (ETEC) must attach to the intestinal mucosa to cause diarrhea in piglets. Prevention of bacterial attachment to the intestinal mucosa is the most effective defense against ETEC-induced diarrhea. Porcine milk fat globule membranes (MFGM) were shown to be able to inhibit attachment of ETEC to the intestinal brush border; however, the specific components of porcine MFGM that inhibited attachment of ETEC to enterocytes were not identified. Accordingly, the purpose of this study was to identify F4ac-binding MFGM proteins by overlay Western blot and affinity chromatography. The proteome of porcine MFGM was characterized and the following F4ac-binding proteins were detected by overlay Western blot and affinity chromatography: lactadherin, butyrophilin, adipophilin, acyl-CoA synthetase 3, and fatty acid-binding protein 3. The biological function of these proteins was not investigated but it is possible that their interaction with F4ac fimbria interferes with bacterial attachment and colonization. PMID:25852227

  12. Role of membrane lipids in peptide hormone function: binding of enkephalins to micelles.

    PubMed Central

    Deber, C M; Behnam, B A

    1984-01-01

    In the course of their biological function, peptide hormones must be transferred from an aqueous phase to the lipid-rich environment of their membrane-bound receptor proteins. We have investigated the possible influence of phospholipids in this process, using 360-MHz 1H and 90-MHz 13C NMR spectroscopy to examine the association of the opioid peptides [Met]- and [Leu]enkephalins (Tyr-Gly-Gly-Phe-Met/Leu) with phospholipid micelles. Binding of peptides to lipid was monitored in NMR spectra by selective chemical shift movements (e.g., the Phe aromatic ring protons) and residue-specific line broadening (e.g., of Met/Leu carbonyl- and alpha-carbon resonances). Results established that the zwitterionic hormones associate hydrophobically both with a neutral lipid (lysophosphatidylcholine) and (also electrostatically) with a negative lipid (lysophosphatidylglycerol). An association constant of Ka = 3.7 X 10(1) M-1 was calculated for the hydrophobic binding of enkephalin to lysophosphatidylcholine. NMR data suggested that enkephalin binds to the lipid with Met/Leu, Phe, and likely Tyr side-chain substituents associated with nonpolar interior regions of the micelle, whereas the COOH-terminal carboxylate moiety of the peptide is located in the surface of the lipid particle. An "attraction-interaction" model is proposed for hormone-lipid association wherein negative lipids attract the hormone electrostatically, while site-specific hydrophobic contacts facilitate its entry, concentration, and orientation into the lipid phase. PMID:6320173

  13. Purification and identification of the fusicoccin binding protein from oat root plasma membrane.

    PubMed

    de Boer, A H; Watson, B A; Cleland, R E

    1989-01-01

    Fusicoccin (FC), a fungal phytotoxin, stimulates the H(+) -ATPase located in the plasma membrane (PM) of higher plants. The first event in the reaction chain leading to enhanced H(+) -efflux seems to be the binding of FC to a FC-binding protein (FCBP) in the PM. We solubilized 90% of the FCBP from oat (Avena sativa L. cv Victory) root PM in an active form with 1% octyl-glucoside. The FCBP was stabilized by the presence of protease inhibitors. The FCBP was purified by affinity chromatography using FC-linked adipic acid dihydrazide agarose (FC-AADA). Upon elution with 8 molar urea, two major protein bands on sodium dodecyl sulfate-polyaerylamide gel electrophoresis with molecular weights of 29,700 and 31,000 were obtained. Successive chromatography on BBAB Bio-Gel A, hexyl agarose, and FC-AADA resulted in the same two bands when the FC-AADA was eluted with sodium dodecyl sulfate. A direct correlation was made between 3H-FC-binding activity and the presence of the two protein bands. The stoichiometry of the 29,700 and 31,000 molecular weight bands was 1:2. This suggests that the FCBP occurs in the native form as a heterotrimer with an apparent molecular weight of approximately 92,000. PMID:11537448

  14. Hepatocellular influx of [14C]oleate reflects membrane transport rather than intracellular metabolism or binding.

    PubMed Central

    Stremmel, W; Berk, P D

    1986-01-01

    When [14C]oleate bound to bovine serum albumin was incubated at 37 degrees C for 7 min with isolated rat hepatocytes in the absence of glucose, the cumulative oleate uptake curve had two components: a rapid, linear segment over the first 30 sec, followed by a slower, curvilinear component. At 173 microM [14C]oleate/albumin (1:1, mol/mol), the initial component had a slope (Vo) of 118 +/- 18 pmol per min per 5 X 10(4) hepatocytes (mean +/- SD). During this initial 30 sec, virtually no oleate was oxidized, and less than 11% was esterified. By 5 min, 79% was esterified; oxidation never exceeded 4%. Addition of 2 mM glucose significantly increased oleate esterification and thereby available oleate binding sites on cytosolic fatty acid binding protein but had no influence on Vo. Pretreatment with trypsin reduced Vo by 49 +/- 15%. These data indicate that the initial component of the oleate uptake curve reflects predominantly influx, whereas the subsequent component reflects a balance between influx, efflux, and intracellular metabolism. Influx is independent of intracellular binding, oxidation, and esterification and may reflect a membrane-associated carrier-mediated process. PMID:3458166

  15. Purification and identification of the fusicoccin binding protein from oat root plasma membrane

    NASA Technical Reports Server (NTRS)

    de Boer, A. H.; Watson, B. A.; Cleland, R. E.

    1989-01-01

    Fusicoccin (FC), a fungal phytotoxin, stimulates the H(+) -ATPase located in the plasma membrane (PM) of higher plants. The first event in the reaction chain leading to enhanced H(+) -efflux seems to be the binding of FC to a FC-binding protein (FCBP) in the PM. We solubilized 90% of the FCBP from oat (Avena sativa L. cv Victory) root PM in an active form with 1% octyl-glucoside. The FCBP was stabilized by the presence of protease inhibitors. The FCBP was purified by affinity chromatography using FC-linked adipic acid dihydrazide agarose (FC-AADA). Upon elution with 8 molar urea, two major protein bands on sodium dodecyl sulfate-polyaerylamide gel electrophoresis with molecular weights of 29,700 and 31,000 were obtained. Successive chromatography on BBAB Bio-Gel A, hexyl agarose, and FC-AADA resulted in the same two bands when the FC-AADA was eluted with sodium dodecyl sulfate. A direct correlation was made between 3H-FC-binding activity and the presence of the two protein bands. The stoichiometry of the 29,700 and 31,000 molecular weight bands was 1:2. This suggests that the FCBP occurs in the native form as a heterotrimer with an apparent molecular weight of approximately 92,000.

  16. FMRFamide: low affinity inhibition of opioid binding to rabbit brain membranes

    SciTech Connect

    Zhu, X.Z.; Raffa, R.B.

    1986-03-05

    FMRFamide (Phe-Met-Arg-Phe-NH/sub 2/) was first isolated from the ganglia of molluscs by Price and Greenberg in 1977. The peptide was subsequently shown to have diverse actions on various types of molluscan and mammalian tissues. The presence of immunoreactive FMRFamide-like material (irFMRF) in multiple areas of rat brain, spinal cord, and gastrointestinal tract suggests that irFMRF may have a physiological role in mammals. Tang, Yang and Costa recently demonstrated that FMRFamide attenuates morphine antinociception in rats and postulated, based on this and several other lines of evidence, that irFMRF might be an endogenous opioid antagonist. In the present study, they tested the ability of FMRFamide to inhibit the binding of opioid receptor ligands to rabbit membrane preparations. FMRFamide inhibited the specific binding of both /sup 3/(H)-dihydromorphine and /sup 3/(H)-ethylketocyclazocine (IC/sub 50/ = 14 ..mu..M and 320 ..mu..M, respectively) in a dose-related manner, suggesting that FMRFamide may affect binding to at least two types of opioid receptors (mu and kappa). These data are consistent with the concept that irFMRF might act as an endogenous opioid antagonist. However, the low affinity of FMRFamide leaves open the possibility of another mechanism of opioid antagonism, such as neuromodulation.

  17. Application of a biotin functionalized QD assay for determining available binding sites on electrospun nanofiber membrane

    PubMed Central

    2011-01-01

    Background The quantification of surface groups attached to non-woven fibers is an important step in developing nanofiber biosensing detection technologies. A method utilizing biotin functionalized quantum dots (QDs) 655 for quantitative analysis of available biotin binding sites within avidin immobilized on electrospun nanofiber membranes was developed. Results A method for quantifying nanofiber bound avidin using biotin functionalized QDs is presented. Avidin was covalently bound to electrospun fibrous polyvinyl chloride (PVC 1.8% COOH w/w containing 10% w/w carbon black) membranes using primary amine reactive EDC-Sulfo NHS linkage chemistry. After a 12 h exposure of the avidin coated membranes to the biotin-QD complex, fluorescence intensity was measured and the total amount of attached QDs was determined from a standard curve of QD in solution (total fluorescence vs. femtomole of QD 655). Additionally, fluorescence confocal microscopy verified the labeling of avidin coated nanofibers with QDs. The developed method was tested against 2.4, 5.2, 7.3 and 13.7 mg spray weights of electrospun nanofiber mats. Of the spray weight samples tested, maximum fluorescence was measured for a weight of 7.3 mg, not at the highest weight of 13.7 mg. The data of total fluorescence from QDs bound to immobilized avidin on increasing weights of nanofiber membrane was best fit with a second order polynomial equation (R2 = .9973) while the standard curve of total fluorescence vs. femtomole QDs in solution had a linear response (R2 = .999). Conclusion A QD assay was developed in this study that provides a direct method for quantifying ligand attachment sites of avidin covalently bound to surfaces. The strong fluorescence signal that is a fundamental characteristic of QDs allows for the measurement of small changes in the amount of these particles in solution or attached to surfaces. PMID:22024374

  18. A specific, high-affinity binding site for the hepta-beta-glucoside elicitor exists in soybean membranes.

    PubMed Central

    Cheong, J J; Hahn, M G

    1991-01-01

    The presence of a specific binding site for a hepta-beta-glucoside elicitor of phytoalexin accumulation has been demonstrated in soybean microsomal membranes. A tyramine conjugate of the elicitor-active hepta-beta-glucoside was prepared and radiolabeled with 125I. The labeled hepta-beta-glucoside-tyramine conjugate was used as a ligand in binding assays with a total membrane fraction prepared from soybean roots. Binding of the radiolabeled hepta-beta-glucoside elicitor was saturable, reversible, and with an affinity (apparent Kd = 7.5 x 10(-10) M) comparable with the concentration of hepta-beta-glucoside required for biological activity. A single class of hepta-beta-glucoside binding sites was found. The binding site was inactivated by proteolysis and by heat treatment, suggesting that the binding site is a protein or glycoprotein. Competitive inhibition of binding of the radiolabeled hepta-beta-glucoside elicitor by a number of structurally related oligoglucosides demonstrated a direct correlation between the binding affinities and the elicitor activities of these oligoglucosides. Thus, the hepta-beta-glucoside-binding protein fulfills criteria expected of a bona fide receptor for the elicitor-active oligosaccharin. PMID:1840905

  19. Characterization of tetanus toxin binding to rat brain membranes. Evidence for a high-affinity proteinase-sensitive receptor.

    PubMed Central

    Pierce, E J; Davison, M D; Parton, R G; Habig, W H; Critchley, D R

    1986-01-01

    Binding of 125I-labelled tetanus toxin to rat brain membranes in 25 mM-Tris/acetate, pH 6.0, was saturable and there was a single class of high-affinity site (KD 0.26-1.14 nM) present in high abundance (Bmax. 0.9-1.89 nmol/mg). The sites were largely resistant to proteolysis and heating but were markedly sensitive to neuraminidase. Trisialogangliosides were effective inhibitors of toxin binding (IC50 10 nM) and trisialogangliosides inserted into membranes lacking a toxin receptor were able to bind toxin with high affinity (KD 2.6 nM). The results are consistent with previous studies and the hypothesis that di- and trisialogangliosides act as the primary receptor for tetanus toxin under these conditions. In contrast, when toxin binding was assayed in Krebs-Ringer buffer, pH 7.4, binding was greatly reduced, was non-saturable and competition binding studies showed evidence for a small number of high-affinity sites (KD 0.42 nM, Bmax. 0.90 pmol/mg) and a larger number of low-affinity sites (KD 146 nM, Bmax. 179 pmol/mg). Treatment of membranes with proteinases, heat, and neuraminidase markedly reduced binding. Trisialogangliosides were poor inhibitors of toxin binding (IC50 11.0 microM), and trisialogangliosides inserted into membranes bound toxin with low affinity. The results suggest that in physiological buffers tetanus toxin binds with high affinity to a protein receptor, and that gangliosides represent only a low-affinity site. Images Fig. 5. PMID:3539106

  20. The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

    PubMed Central

    Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A.; Korn, Edward D.

    2014-01-01

    F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave. PMID:24747353

  1. Sex bias in CNS autoimmune disease mediated by androgen control of autoimmune regulator.

    PubMed

    Zhu, Meng-Lei; Bakhru, Pearl; Conley, Bridget; Nelson, Jennifer S; Free, Meghan; Martin, Aaron; Starmer, Joshua; Wilson, Elizabeth M; Su, Maureen A

    2016-01-01

    Male gender is protective against multiple sclerosis and other T-cell-mediated autoimmune diseases. This protection may be due, in part, to higher androgen levels in males. Androgen binds to the androgen receptor (AR) to regulate gene expression, but how androgen protects against autoimmunity is not well understood. Autoimmune regulator (Aire) prevents autoimmunity by promoting self-antigen expression in medullary thymic epithelial cells, such that developing T cells that recognize these self-antigens within the thymus undergo clonal deletion. Here we show that androgen upregulates Aire-mediated thymic tolerance to protect against autoimmunity. Androgen recruits AR to Aire promoter regions, with consequent enhancement of Aire transcription. In mice and humans, thymic Aire expression is higher in males compared with females. Androgen administration and male gender protect against autoimmunity in a multiple sclerosis mouse model in an Aire-dependent manner. Thus, androgen control of an intrathymic Aire-mediated tolerance mechanism contributes to gender differences in autoimmunity. PMID:27072778

  2. Characterization of the proton binding sites of extracellular polymeric substances in an anaerobic membrane bioreactor.

    PubMed

    Liu, Yi; Chang, Sheng; Defersha, Fantahun M

    2015-07-01

    This paper focuses on the characterization of the chemical compositions and acidic constants of the extracellular polymeric substances (EPSs) in an anaerobic membrane bioreactor treating synthetic brewery wastewater by using chemical analysis, linear programming analysis (LPA) of titration data, and FT-IR analysis. The linear programming analysis of titration data revealed that the EPSs have proton binding sites with pKa values from pKa ≤ 6, between 6 and 7, and approximately 9.8. The strong acidic sites (pKa ≤ 6) and some weak acidic sites (7.5 < pKa < 9.0) were found to be readily removed by 0.45-μm membrane filtration. In addition, the FT-IR analysis confirmed the presence of proteins, carbohydrates, nucleic acids, and lipids in the EPS samples. Based on the FT-IR analysis and the main chemical functional groups at the bacterial cell surfaces, the identified proton binding sites were related to carboxyl, phosphate, and hydroxyl/amine groups with pKa values of 4.6 ± 0.7, 6.6 ± 0.01, and 9.7 ± 0.1, respectively, with the corresponding respective intensities of 0.31 ± 0.05, 0.96 ± 0.3, and 1.53 ± 0.3 mmole/g-EPS. The pKa values and intensities of the proton binding sites are the fundamental molecular properties of EPSs that affect the EPS charge, molecular interactions, and metal complexation characteristics. Determination of such properties can advance Derjaguin-Landau-Verwey-Overbeek (DLVO)-based concentration polarization modeling, facilitate the estimation of the osmotic pressure of the EPS concentration polarization layers, and lead to a deeper understanding of the role of metal complexation in membrane fouling. PMID:25935368

  3. High Membrane Curvature Enhances Binding, Conformational Changes, and Fibrillation of Amyloid-β on Lipid Bilayer Surfaces.

    PubMed

    Sugiura, Yuuki; Ikeda, Keisuke; Nakano, Minoru

    2015-10-27

    Aggregation of the amyloid-β (Aβ) protein and the formation of toxic aggregates are the possible pathogenic pathways in Alzheimer's disease. Accumulating evidence suggests that lipid membranes play key roles in protein aggregation, although the intermolecular forces that drive the interactions between Aβ-(1-40) and the membranes vary in different membrane systems. Here, we observed that a high positive curvature of lipid vesicles with diameters of ∼30 nm enhanced the association of Aβ with anionic phosphatidylglycerol membranes in the liquid-crystalline phase and with zwitterionic phosphatidylcholine membranes in the gel phase. The binding modes of Aβ to these membranes differ in terms of the location of the protein on the membrane and of the protein secondary structure. The fibrillation of Aβ was accelerated in the presence of the vesicles and at high protein-to-lipid ratios. Under these conditions, the protein accumulated on the surfaces, as demonstrated by a high (10(7) M(-1)) binding constant. Our findings suggest that packing defects on membranes with high curvatures, such as the intraluminal vesicles in multivesicular bodies and the exosomes, might result in the accumulation of toxic protein aggregates. PMID:26474149

  4. Glycosylation is essential for translocation of carp retinol-binding protein across the endoplasmic reticulum membrane

    SciTech Connect

    Devirgiliis, Chiara; Gaetani, Sancia; Apreda, Marianna; Bellovino, Diana . E-mail: bellovino@inran.it

    2005-07-01

    Retinoid transport is well characterized in many vertebrates, while it is still largely unexplored in fish. To study the transport and utilization of vitamin A in these organisms, we have isolated from a carp liver cDNA library retinol-binding protein, its plasma carrier. The primary structure of carp retinol-binding protein is very conserved, but presents unique features compared to those of the correspondent proteins isolated and characterized so far in other species: it has an uncleavable signal peptide and two N-glycosylation sites in the NH{sub 2}-terminal region of the protein that are glycosylated in vivo. In this paper, we have investigated the function of the carbohydrate chains, by constructing three mutants deprived of the first, the second or both carbohydrates. The results of transient transfection of wild type and mutant retinol-binding protein in Cos cells followed by Western blotting and immunofluorescence analysis have shown that the absence of both carbohydrate moieties blocks secretion, while the presence of one carbohydrate group leads to an inefficient secretion. Experiments of carp RBP mRNA in vitro translation in a reticulocyte cell-free system in the presence of microsomes have demonstrated that N-glycosylation is necessary for efficient translocation across the endoplasmic reticulum membranes. Moreover, when Cos cells were transiently transfected with wild type and mutant retinol-binding protein (aa 1-67)-green fluorescent protein fusion constructs and semi-permeabilized with streptolysin O, immunofluorescence analysis with anti-green fluorescent protein antibody revealed that the double mutant is exposed to the cytosol, thus confirming the importance of glycan moieties in the translocation process.

  5. Computing membrane-AQP5-phosphatidylserine binding affinities with hybrid steered molecular dynamics approach

    PubMed Central

    Chen, Liao Y

    2015-01-01

    In order to elucidate how phosphatidylserine (PS6) interacts with AQP5 in a cell membrane, we develop a hybrid steered molecular dynamics (hSMD) method that involves (1) simultaneously steering two centers of mass of two selected segments of the ligand and (2) equilibrating the ligand-protein complex with and without biasing the system. Validating hSMD, we first study vascular endothelial growth factor receptor 1 (VEGFR1) in complex with N-(4-Chlorophenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (8ST), for which the binding energy is known from in vitro experiments. In this study, our computed binding energy well agrees with the experimental value. Knowing the accuracy of this hSMD method, we apply it to the AQP5-lipid-bilayer system to answer an outstanding question relevant to AQP5’s physiological function: Will the PS6, a lipid having a single long hydrocarbon tail that was found in the central pore of the AQP5 tetramer crystal, actually bind to and inhibit AQP5’s central pore under near-physiological conditions, namely, when AQP5 tetramer is embedded in a lipid bilayer? We find, in silico, using the CHARMM 36 force field, that binding PS6 to AQP5 is a factor of 3 million weaker than “binding” it in the lipid bilayer. This suggests that AQP5’s central pore will not be inhibited by PS6 or a similar lipid in a physiological environment. PMID:25955791

  6. The 43-K protein, v1, associated with acetylcholine receptor containing membrane fragments is an actin-binding protein.

    PubMed Central

    Walker, J H; Boustead, C M; Witzemann, V

    1984-01-01

    Acetylcholine receptor enriched membrane fragments were obtained from the electric organs of Torpedo marmorata. The purified membrane fragments contained several proteins in addition to the acetylcholine receptor subunits. One of these was shown to be actin by means of immune blotting with a monoclonal antibody. Brief treatment of the membranes with pH 11.0 buffer removed actin and the other non-receptor proteins including the receptor-associated 43 000 mol. wt. polypeptide. This polypeptide was shown to bind actin after transferring the proteins from one- and two-dimensional polyacrylamide gels to nitrocellulose paper and incubating the nitrocellulose blots with actin. Specifically bound actin was demonstrated using the monoclonal antibodies to actin. No calcium or calmodulin dependency of binding was observed. The findings suggest that the 43 000 mol. wt. polypeptide is a link between the membrane-bound acetylcholine receptor and the cytoskeleton. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6389118

  7. Androgen receptor-mediated non-genomic regulation of prostate cancer cell proliferation

    PubMed Central

    Liao, Ross S.; Ma, Shihong; Miao, Lu; Li, Rui; Yin, Yi

    2013-01-01

    Androgen receptor (AR)-mediated signaling is necessary for prostate cancer cell proliferation and an important target for therapeutic drug development. Canonically, AR signals through a genomic or transcriptional pathway, involving the translocation of androgen-bound AR to the nucleus, its binding to cognate androgen response elements on promoter, with ensuing modulation of target gene expression, leading to cell proliferation. However, prostate cancer cells can show dose-dependent proliferation responses to androgen within minutes, without the need for genomic AR signaling. This proliferation response known as the non-genomic AR signaling is mediated by cytoplasmic AR, which facilitates the activation of kinase-signaling cascades, including the Ras-Raf-1, phosphatidyl-inositol 3-kinase (PI3K)/Akt and protein kinase C (PKC), which in turn converge on mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) activation, leading to cell proliferation. Further, since activated ERK may also phosphorylate AR and its coactivators, the non-genomic AR signaling may enhance AR genomic activity. Non-genomic AR signaling may occur in an ERK-independent manner, via activation of mammalian target of rapamycin (mTOR) pathway, or modulation of intracellular Ca2+ concentration through plasma membrane G protein-coupled receptors (GPCRs). These data suggest that therapeutic strategies aimed at preventing AR nuclear translocation and genomic AR signaling alone may not completely abrogate AR signaling. Thus, elucidation of mechanisms that underlie non-genomic AR signaling may identify potential mechanisms of resistance to current anti-androgens and help developing novel therapies that abolish all AR signaling in prostate cancer. PMID:26816736

  8. TRPM8 channel as a novel molecular target in androgen-regulated prostate cancer cells.

    PubMed

    Asuthkar, Swapna; Velpula, Kiran Kumar; Elustondo, Pia A; Demirkhanyan, Lusine; Zakharian, Eleonora

    2015-07-10

    The cold and menthol receptor TRPM8 is highly expressed in prostate and prostate cancer (PC). Recently, we identified that TRPM8 is as an ionotropic testosterone receptor. The TRPM8 mRNA is expressed in early prostate tumors with high androgen levels, while anti-androgen therapy greatly reduces its expression. Here, from the chromatin-immunoprecipitation (ChIP) analysis, we found that an androgen response element (ARE) mediates androgen regulation of trpm8. Furthermore, using immunofluorescence, calcium-imaging and planar lipid bilayers, we identified that TRPM8 channel is functionally regulated by androgens in the prostate. Although TRPM8 mRNA is expressed at high levels, we found that the TRPM8 protein undergoes ubiquitination and degradation in PC cells. The mass-spectrometry analysis of TRPM8, immunoprecipitated from LNCaP cells identified ubiquitin-like modifier-activating enzyme 1 (UBA1). PYR-41, a potent inhibitor of initial enzyme in the ubiquitination cascade, UBA1, increased TRPM8 activity on the plasma membrane (PM) of LNCaP cells. Furthermore, PYR-41-mediated PMTRPM8 activity was accompanied by enhanced activation of p53 and Caspase-9. Interestingly, we found that the trpm8 promoter possesses putative binding sites for p53 and that the overexpression of p53 increased the TRPM8 mRNA levels. In addition to the genomic regulation of TRPM8 by AR and p53, our findings indicate that the testosterone-induced PMTRPM8 activity elicits Ca2+ uptake, subsequently causing apoptotic cell death. These findings support the strategy of rescuing PMTRPM8 expression as a new therapeutic application through the regulation of PC cell growth and proliferation. PMID:25980497

  9. TRPM8 channel as a novel molecular target in androgen-regulated prostate cancer cells

    PubMed Central

    Asuthkar, Swapna; Velpula, Kiran Kumar; Elustondo, Pia A.; Demirkhanyan, Lusine; Zakharian, Eleonora

    2015-01-01

    The cold and menthol receptor TRPM8 is highly expressed in prostate and prostate cancer (PC). Recently, we identified that TRPM8 is as an ionotropic testosterone receptor. The TRPM8 mRNA is expressed in early prostate tumors with high androgen levels, while anti-androgen therapy greatly reduces its expression. Here, from the chromatin-immunoprecipitation (ChIP) analysis, we found that an androgen response element (ARE) mediates androgen regulation of trpm8. Furthermore, using immunofluorescence, calcium-imaging and planar lipid bilayers, we identified that TRPM8 channel is functionally regulated by androgens in the prostate. Although TRPM8 mRNA is expressed at high levels, we found that the TRPM8 protein undergoes ubiquitination and degradation in PC cells. The mass-spectrometry analysis of TRPM8, immunoprecipitated from LNCaP cells identified ubiquitin-like modifier-activating enzyme 1 (UBA1). PYR-41, a potent inhibitor of initial enzyme in the ubiquitination cascade, UBA1, increased TRPM8 activity on the plasma membrane (PM) of LNCaP cells. Furthermore, PYR-41-mediated PMTRPM8 activity was accompanied by enhanced activation of p53 and Caspase-9. Interestingly, we found that the trpm8 promoter possesses putative binding sites for p53 and that the overexpression of p53 increased the TRPM8 mRNA levels. In addition to the genomic regulation of TRPM8 by AR and p53, our findings indicate that the testosterone-induced PMTRPM8 activity elicits Ca2+ uptake, subsequently causing apoptotic cell death. These findings support the strategy of rescuing PMTRPM8 expression as a new therapeutic application through the regulation of PC cell growth and proliferation. PMID:25980497

  10. Granule membrane protein 140 (GMP140) binds to carcinomas and carcinoma-derived cell lines.

    PubMed Central

    Aruffo, A; Dietsch, M T; Wan, H; Hellström, K E; Hellström, I

    1992-01-01

    The glycoproteins granule membrane protein 140 (GMP140), endothelial-leukocyte adhesion molecule 1 (ELAM-1), and Leu-8 are members of a family of glycoprotein receptors (selectins or LEC-CAMs) that play an important role in adhesive interactions between circulating leukocytes and vascular endothelium. Recently it has been reported that ELAM-1 is able to mediate the binding of the colon carcinoma cell line HT-29 to cytokine-activated vascular endothelium, suggesting that tumor cell adhesion to vascular endothelium, a prerequisite for tumor extravasation and metastasis, is in part the result of adhesive interactions between blood-borne tumor cells and cell surface proteins expressed by vascular endothelium. Here, using an approach in which soluble immunoglobulin chimeras of the GMP140 and ELAM-1 receptors were prepared and used to carry out immunohistological studies, we establish that GMP140 binds to tumor cells in a variety of human carcinoma tissue sections (colon, lung, and breast), whereas ELAM-1 binds exclusively to tumor cells in colon carcinoma tissue sections. In addition, GMP140 was found to bind to the cell surface of a number of cell lines derived from various carcinomas but not from melanomas, whereas ELAM-1 bound only colon carcinoma cell lines. We further investigated the nature of the ligands of GMP140 and ELAM-1 on the surface of the carcinoma cells and found that the GMP140 ligand on the surface of tumor cells appears to be distinct from that expressed on the myeloid cell line HL-60. Neuraminidase treatment of a breast carcinoma cell line does not affect, or in some instances increases, GMP140 binding, whereas it completely abolishes GMP140 binding to HL-60 cells. On the other hand, the ligand of ELAM-1 on both the colon carcinoma and HL-60 cells is neuraminidase sensitive in accord with its identification as sialyl-CD15. Parallel results were obtained with neuraminidase-treated frozen carcinoma tissue sections. The present findings form the basis

  11. Membrane Binding Events in the Initiation and Propagation Phases of Tissue Factor-initiated Zymogen Activation under Flow*

    PubMed Central

    Haynes, Laura M.; Dubief, Yves C.; Mann, Kenneth G.

    2012-01-01

    This study investigates the dynamics of zymogen activation when both extrinsic tenase and prothrombinase are assembled on an appropriate membrane. Although the activation of prothrombin by surface-localized prothrombinase is clearly mediated by flow-induced dilutional effects, we find that when factor X is activated in isolation by surface-localized extrinsic tenase, it exhibits characteristics of diffusion-mediated activation in which diffusion of substrate to the catalytically active region is rate-limiting. When prothrombin and factor X are activated coincident with each other, competition for available membrane binding sites masks the diffusion-limiting effects of factor X activation. To verify the role of membrane binding in the activation of factor X by extrinsic tenase under flow conditions, we demonstrate that bovine lactadherin competes for both factor X and Xa binding sites, limiting factor X activation and forcing the release of bound factor Xa from the membrane at a venous shear rate (100 s−1). Finally, we present steady-state models of prothrombin and factor X activation under flow showing that zymogen and enzyme membrane binding events further regulate the coagulation process in an open system representative of the vasculature geometry. PMID:22187432

  12. Membrane texture induced by specific protein binding and receptor clustering: active roles for lipids in cellular function.

    PubMed

    Watkins, E B; Miller, C E; Majewski, J; Kuhl, T L

    2011-04-26

    Biological membranes are complex, self-organized structures that define boundaries and compartmentalize space in living matter. Composed of a wide variety of lipid and protein molecules, these responsive surfaces mediate transmembrane signaling and material transport within the cell and with its environment. It is well known that lipid membrane properties change as a function of composition and phase state, and that protein-lipid interactions can induce changes in the membrane's properties and biochemical response. Here, molecular level changes in lipid organization induced by multivalent toxin binding were investigated using grazing incidence X-ray diffraction. Structural changes to lipid monolayers at the air-water interface and bilayers at the solid-water interface were studied before and after specific binding of cholera toxin to membrane embedded receptors. At biologically relevant surface pressures, protein binding perturbed lipid packing within monolayers and bilayers resulting in topological defects and the emergence of a new orientationally textured lipid phase. In bilayers this altered lipid order was transmitted from the receptor laden exterior membrane leaflet to the inner leaflet, representing a potential mechanism for lipid mediated outside-in signaling by multivalent protein binding. It is further hypothesized that cell-surface micro-domains exhibiting this type of lipid order may serve as nucleation sites for vesicle formation in clathrin independent endocytosis of cholera toxin. PMID:21474780

  13. Parkinson’s Protein α-Synuclein Binds Efficiently and with a Novel Conformation to Two Natural Membrane Mimics

    PubMed Central

    Kumar, Pravin; Segers-Nolten, Ine M. J.; Schilderink, Nathalie; Subramaniam, Vinod; Huber, Martina

    2015-01-01

    Binding of human α-Synuclein, a protein associated with Parkinson’s disease, to natural membranes is thought to be crucial in relation to its pathological and physiological function. Here the binding of αS to small unilamellar vesicles mimicking the inner mitochondrial and the neuronal plasma membrane is studied in situ by continuous wave and pulsed electron paramagnetic resonance. Local binding information of αS spin labeled by MTSL at positions 56 and 69 respectively shows that also helix 2 (residues 50–100) binds firmly to both membranes. By double electron-electron resonance (DEER) on the mutant spin labeled at positions 27 and 56 (αS 27/56) a new conformation on the membrane is found with a distance of 3.6 nm/ 3.7 nm between residues 27 and 56. In view of the low negative charge density of these membranes, the strong interaction is surprising, emphasizing that function and pathology of αS could involve synaptic vesicles and mitochondria. PMID:26588454

  14. Binding of /sup 3/H-diazepam with brain synaptic membranes during the development of generalized epileptic activity

    SciTech Connect

    Borydukov, M.M.; Bogdanova, E.D.; Kryzhanovskii, G.N.; Nikushin, E.V.; Prilipko, L.L.

    1986-04-01

    The authors study the effect of the development of generalized epileptic activity in the rat cerebral cortex on binding of tritium-diazepam with synaptic membranes. The experiments showed that dependence of the quantity of tritium-diazepam bound with synaptic membranes on the concentration of label in the incubation medium is linear in character on a Scatchard plot. This is evidence of the existence of only one type of binding site withK /SUB d/ = 4.40 plus or minus 0.15 nM and B /SUB max/ = 410 plus or minus 35 fm fmoles/mg protein.

  15. Effect of lithocholic acid feeding on plasma lipoproteins and binding of radioiodinated human lipoproteins to hepatic membranes in rats.

    PubMed

    Loo, G; Kessie, G; Berlin, E; Nair, P P

    1992-06-01

    1. Male Sprague-Dawley rats fed diets containing 0.25% lithocholic acid for 6 weeks exhibited elevated serum cholesterol. 2. The rats were fed diets containing 5 or 20% fat with and without the lithocholate and/or oxytetracycline-HCl. 3. The cholesterol elevation was associated with high density lipoprotein (HDL) and not very low density lipoprotein (VLDL) or low density lipoprotein (LDL). 4. Specific binding of human [125I]HDL to hepatic membranes was lowered in lithocholate-fed rats, but binding of human [125I]LDL to these membranes was not affected. PMID:1354585

  16. Active transport of maltose in membrane vesicles obtained from Escherichia coli cells producing tethered maltose-binding protein.

    PubMed Central

    Dean, D A; Fikes, J D; Gehring, K; Bassford, P J; Nikaido, H

    1989-01-01

    Attempts to reconstitute periplasmic binding protein-dependent transport activity in membrane vesicles have often resulted in systems with poor and rather inconsistent activity, possibly because of the need to add a large excess of purified binding protein to the vesicles. We circumvented this difficulty by using a mutant which produces a precursor maltose-binding protein that is translocated across the cytoplasmic membrane but is not cleaved by the signal peptidase (J. D. Fikes and P. J. Bassford, Jr., J. Bacteriol. 169:2352-2359, 1987). The protein remains tethered to the cytoplasmic membrane, presumably through the hydrophobic signal sequence, and we show here that the spheroplasts and membrane vesicles prepared from this mutant catalyze active maltose transport without the addition of purified maltose-binding protein. In vesicles, the transport requires electron donors, such as ascorbate and phenazine methosulfate or D-lactate. However, inhibition by dicyclohexylcarbodiimide and stimulation of transport by the inculsion of ADP or ATP in the intravesicular space suggest that ATP (or compounds derived from it) is involved in the energization of the transport. The transport activity of intact cells can be recovered without much inactivation in the vesicles, and their high activity and ease of preparation will be useful in studies of the mechanism of the binding protein-dependent transport process. Images PMID:2644203

  17. C1q binding and activation of the complement classical pathway by Klebsiella pneumoniae outer membrane proteins.

    PubMed Central

    Albertí, S; Marqués, G; Camprubí, S; Merino, S; Tomás, J M; Vivanco, F; Benedí, V J

    1993-01-01

    The mechanisms of killing of Klebsiella pneumoniae serum-sensitive strains in nonimmune serum by the complement classical pathway have been studied. The bacterial cell surface components that bind C1q more efficiently were identified as two major outer membrane proteins, presumably the porins of this bacterial species. These two outer membrane proteins were isolated from a representative serum-sensitive strain. We have demonstrated that in their purified form, they bind C1q and activate the classical pathway in an antibody-independent manner, with the subsequent consumption of C4 and reduction of the serum total hemolytic activity. Activation of the classical pathway has been observed in human nonimmune serum and agammaglobulinemic serum (both depleted in factor D). Binding of C1q to other components of the bacterial outer membrane, in particular the rough lipopolysaccharide, could not be demonstrated. Activation of the classical pathway by this lipopolysaccharide was also much less efficient than activation by the two outer membrane proteins. The antibody-independent binding of C1q to serum-sensitive strains was independent of the presence of capsular polysaccharide, while strains possessing lipopolysaccharide O antigen bind less C1q and are resistant to complement-mediated killing. Images PMID:8432605

  18. Early Events in Chikungunya Virus Infection—From Virus Cell Binding to Membrane Fusion

    PubMed Central

    van Duijl-Richter, Mareike K. S.; Hoornweg, Tabitha E.; Rodenhuis-Zybert, Izabela A.; Smit, Jolanda M.

    2015-01-01

    Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne alphavirus causing millions of infections in the tropical and subtropical regions of the world. CHIKV infection often leads to an acute self-limited febrile illness with debilitating myalgia and arthralgia. A potential long-term complication of CHIKV infection is severe joint pain, which can last for months to years. There are no vaccines or specific therapeutics available to prevent or treat infection. This review describes the critical steps in CHIKV cell entry. We summarize the latest studies on the virus-cell tropism, virus-receptor binding, internalization, membrane fusion and review the molecules and compounds that have been described to interfere with virus cell entry. The aim of the review is to give the reader a state-of-the-art overview on CHIKV cell entry and to provide an outlook on potential new avenues in CHIKV research. PMID:26198242

  19. Different binding of stimulatory-type and blocking-type TSH receptor antibody with guinea-pig testis membrane.

    PubMed

    Inui, T; Ochi, Y; Hachiya, T; Chen, W; Nakajima, Y; Kajita, Y; Ogura, H

    1991-11-01

    A receptor assay using [125I]bTSH-binding to guinea-pig testis membrane was developed. Unlabelled hCG and FSH inhibited [125I]bTSH binding. In patients with Graves' disease and in untreated hyperthyroid patients, almost all long-acting thyroid stimulators and thyroid-stimulating antibodies, respectively did not inhibit [125I]bTSH binding, which on the other hand was inhibited by thyroid stimulation blocking antibodies in patients with primary hypothyroidism. When the inhibitory effect on the binding of [125I]hCG and 125I-synthetic alpha-subunit peptide (alpha 26-46) of hCG to testis membrane was examined, bTSH resulted in a significant inhibition. However, all three kinds of TSH receptor antibodies had no inhibitory effect. This study demonstrated 1. interaction of alpha-subunit of TSH and hCG with the testicular receptor; 2. binding of thyroid stimulation-blocking antibody and lack of binding of thyroid-stimulating antibody to the testicular TSH receptor in spite of binding of these TSH receptor antibodies to the thyroidal TSH receptor, and 3. lack of binding of thyroid-stimulating antibody and thyroid stimulation-blocking antibody to the testicular gonadotropin receptor. PMID:1684686

  20. Ligand binding to the ACBD6 protein regulates the acyl-CoA transferase reactions in membranes.

    PubMed

    Soupene, Eric; Kuypers, Frans A

    2015-10-01

    The binding determinants of the human acyl-CoA binding domain-containing protein (ACBD) 6 and its function in lipid renewal of membranes were investigated. ACBD6 binds acyl-CoAs of a chain length of 6 to 20 carbons. The stoichiometry of the association could not be fitted to a 1-to-1 model. Saturation of ACBD6 by C16:0-CoA required higher concentration than less abundant acyl-CoAs. In contrast to ACBD1 and ACBD3, ligand binding did not result in the dimerization of ACBD6. The presence of fatty acids affected the binding of C18:1-CoA to ACBD6, dependent on the length, the degree of unsaturation, and the stereoisomeric conformation of their aliphatic chain. ACBD1 and ACBD6 negatively affected the formation of phosphatidylcholine (PC) and phosphatidylethanolamine in the red blood cell membrane. The acylation rate of lysophosphatidylcholine into PC catalyzed by the red cell lysophosphatidylcholine-acyltransferase 1 protein was limited by the transfer of the acyl-CoA substrate from ACBD6 to the acyltransferase enzyme. These findings provide evidence that the binding properties of ACBD6 are adapted to prevent its constant saturation by the very abundant C16:0-CoA and protect membrane systems from the detergent nature of free acyl-CoAs by controlling their release to acyl-CoA-utilizing enzymes. PMID:26290611

  1. A fullerene C60-based ligand in a stationary phase for affine chromatography of membrane porphyrin-binding proteins

    NASA Astrophysics Data System (ADS)

    Amirshakhi, N.; Alyautdin, R. N.; Orlov, A. P.; Poloznikov, A. A.; Kuznetsov, D. A.

    2008-11-01

    A new affine chromatography technique is suggested for the purification of porphyrin-binding proteins (PBP) from mammal cell membranes. The procedure uses new fullerene-porphyrin ligands immobilized on agarose and bound to the polysaccharide matrix via the epoxycyclohexyl residue. A selective PBP stationary phase was used in a single-column chromatography run for the complete purification of a monomeric protein (17.6 kDa) from mitochondrial membranes of rat myocardium. This protein was characterized by high affinity for porphyrin-related structures. To separate it from other nonspecifically sorbed membrane proteins, synchronous linear pH and ionic strength gradients were used.

  2. AAA ATPases regulate membrane association of yeast oxysterol binding proteins and sterol metabolism.

    PubMed

    Wang, Penghua; Zhang, Yong; Li, Hongzhe; Chieu, Hai Kee; Munn, Alan L; Yang, Hongyuan

    2005-09-01

    The yeast genome encodes seven oxysterol binding protein homologs, Osh1p-Osh7p, which have been implicated in regulating intracellular lipid and vesicular transport. Here, we show that both Osh6p and Osh7p interact with Vps4p, a member of the AAA (ATPases associated with a variety of cellular activities) family. The coiled-coil domain of Osh7p was found to interact with Vps4p in a yeast two-hybrid screen and the interaction between Osh7p and Vps4p appears to be regulated by ergosterol. Deletion of VPS4 induced a dramatic increase in the membrane-associated pools of Osh6p and Osh7p and also caused a decrease in sterol esterification, which was suppressed by overexpression of OSH7. Lastly, overexpression of the coiled-coil domain of Osh7p (Osh7pCC) resulted in a multivesicular body sorting defect, suggesting a dominant negative role of Osh7pCC possibly through inhibiting Vps4p function. Our data suggest that a common mechanism may exist for AAA proteins to regulate the membrane association of yeast OSBP proteins and that these two protein families may function together to control subcellular lipid transport. PMID:16096648

  3. Ribosome binding sites visualized on freeze-fractured membranes of the rough endoplasmic reticulum.

    PubMed

    Giddings, T H; Staehelin, L A

    1980-04-01

    Freeze-fracture micrographs of cells of the green alga Micrasterias denticulata stabilized by ultrarapid freezing reveal imprints of polysomes on the rough endoplasmic reticulum membranes. The imprints appear as broad, spiral ridges on the P faces and as corresponding wide grooves on the E faces of the membranes. Distinct 110-A particles with a spacing of 270 +/- 45 A are associated with the P-face ridges. Where imprints of individual ribosomes can be discerned, it is seen that there is a 1:1 relationship between the ribosomes and the 110-A particles, and that the 110-A particles are located in a peripheral position with respect to the polysome spirals. We propose that the 110-A particles could be structural equivalents of ribosome-binding sites, consisting of a molecule each of ribophorins I and II and a nascent polypeptide chain. These observations suggest that the spiral form of polysomes could result from the forces generated by the extrusion of the growing polypeptide chains to one side of the polysome. PMID:7364870

  4. Ribosome binding sites visualized on freeze-fractured membranes of the rough endoplasmic reticulum

    PubMed Central

    1980-01-01

    Freeze-fracture micrographs of cells of the green alga Micrasterias denticulata stabilized by ultrarapid freezing reveal imprints of polysomes on the rough endoplasmic reticulum membranes. The imprints appear as broad, spiral ridges on the P faces and as corresponding wide grooves on the E faces of the membranes. Distinct 110-A particles with a spacing of 270 +/- 45 A are associated with the P-face ridges. Where imprints of individual ribosomes can be discerned, it is seen that there is a 1:1 relationship between the ribosomes and the 110-A particles, and that the 110-A particles are located in a peripheral position with respect to the polysome spirals. We propose that the 110- A particles could be structural equivalents of ribosome-binding sites, consisting of a molecule each of ribophorins I and II and a nascent polypeptide chain. These observations suggest that the spiral form of polysomes could result from the forces generated by the extrusion of the growing polypeptide chains to one side of the polysome. PMID:7364870

  5. The dynamin-binding domains of Dap160/intersectin affect bulk membrane retrieval in synapses

    PubMed Central

    Winther, Åsa M. E.; Jiao, Wei; Vorontsova, Olga; Rees, Kathryn A.; Koh, Tong-Wey; Sopova, Elena; Schulze, Karen L.; Bellen, Hugo J.; Shupliakov, Oleg

    2013-01-01

    Summary Dynamin-associated protein 160 kDa (Dap160)/intersectin interacts with several synaptic proteins and affects endocytosis and synapse development. The functional role of the different protein interaction domains is not well understood. Here we show that Drosophila Dap160 lacking the dynamin-binding SH3 domains does not affect the development of the neuromuscular junction but plays a key role in synaptic vesicle recycling. dap160 mutants lacking dynamin-interacting domains no longer accumulate dynamin properly at the periactive zone, and it becomes dispersed in the bouton during stimulation. This is accompanied by a reduction in uptake of the dye FM1-43 and an accumulation of large vesicles and membrane invaginations. However, we do not observe an increase in the number of clathrin-coated intermediates. We also note a depression in evoked excitatory junction potentials (EJPs) during high-rate stimulation, accompanied by aberrantly large miniature EJPs. The data reveal the important role of Dap160 in the targeting of dynamin to the periactive zone, where it is required to suppress bulk synaptic vesicle membrane retrieval during high-frequency activity. PMID:23321638

  6. Characterization of the ribosomal binding site in rat liver rough microsomes: ribophorins I and II, two integral membrane proteins related to ribosome binding.

    PubMed

    Kreibich, G; Czakó-Graham, M; Grebenau, R; Mok, W; Rodriguez-Boulan, E; Sabatini, D D

    1978-01-01

    Rat liver rough endoplasmic reticulum membranes (ER) contain two characteristic transmembrane glycoproteins which have been designated ribophorins I and II and are absent from smooth ER membranes. These proteins (MW 65,000 and 63,000 respectively) are related to the binding sites for ribosomes, as suggested by the following findings: i) The ribophorin content of the rough ER membranes corresponds stoichiometrically to the number of bound ribosomes; ii) ribophorins are quantitatively recovered with the bound polysomes after most other ER membrane proteins are dissolved with the nonionic detergent Kyro EOB; iii) in intact rough microsomes ribophorins can be cross-linked chemically to the ribosomes and therefore are in close proximity to them. Treatment of rough microsomes with a low Triton-X-100 concentration leads to the lateral displacement of ribosomes on the microsomal surface and to the formation of aggregates of bound ribosomes in areas of membranes which frequently invaginate into the microsomal lumen. Subfractionation of Triton-treated microsomes containing invaginations led to the recovery of smooth and "rough-inverted" vesicles. Ribophorins were present only in the latter fraction, indicating that both proteins are displaced together with the ribosomes when these aggregate without detaching. Measurements of the ribosome-binding capacity of rough and smooth microsomal membranes reconstituted after solubilization with detergents suggest that ribophorins are necessary for in vitro ribosome binding. Ribophorin-like proteins were found in rough microsomes obtained from secretory tissues of several animal species. The two proteins present in rat lacrimal gland microsomes have the same mobility as hepatocyte ribophorins and cross-react with antisera against them. PMID:723266

  7. Membranes serve as allosteric activators of phospholipase A2, enabling it to extract, bind, and hydrolyze phospholipid substrates

    PubMed Central

    Mouchlis, Varnavas D.; Bucher, Denis; McCammon, J. Andrew; Dennis, Edward A.

    2015-01-01

    Defining the molecular details and consequences of the association of water-soluble proteins with membranes is fundamental to understanding protein–lipid interactions and membrane functioning. Phospholipase A2 (PLA2) enzymes, which catalyze the hydrolysis of phospholipid substrates that compose the membrane bilayers, provide the ideal system for studying protein–lipid interactions. Our study focuses on understanding the catalytic cycle of two different human PLA2s: the cytosolic Group IVA cPLA2 and calcium-independent Group VIA iPLA2. Computer-aided techniques guided by deuterium exchange mass spectrometry data, were used to create structural complexes of each enzyme with a single phospholipid substrate molecule, whereas the substrate extraction process was studied using steered molecular dynamics simulations. Molecular dynamic simulations of the enzyme–substrate–membrane systems revealed important information about the mechanisms by which these enzymes associate with the membrane and then extract and bind their phospholipid substrate. Our data support the hypothesis that the membrane acts as an allosteric ligand that binds at the allosteric site of the enzyme’s interfacial surface, shifting its conformation from a closed (inactive) state in water to an open (active) state at the membrane interface. PMID:25624474

  8. Binding of Streptococcus mutans antigens to heart and kidney basement membranes.

    PubMed Central

    Stinson, M W; Barua, P K; Bergey, E J; Nisengard, R J; Neiders, M E; Albini, B

    1984-01-01

    Using indirect immunofluorescence, alkali-extracted components of Streptococcus mutans were found to bind in vitro to capillary walls and sarcolemmal sheaths of monkey cardiac muscle and to glomerular and tubular basement membranes of monkey kidney. Adsorption of S. mutans components to tissue fragments was also detected by indirect radioimmunoassay and immunoblotting on nitrocellulose paper. Antibodies did not bind to untreated, control tissues in these experiments, proving that antigens shared by S. mutans and tissue components were not involved. Rabbit and monkey heart and kidney components bound S. mutans antigens of 24,000, 35,000, and 65,000 Mr. Monkey heart also bound molecules of 90,000 and 120,000 Mr. Rabbits immunized by intravenous injection of disrupted S. mutans cells developed severe nephritis that was characterized by the deposition of immunoglobulins, complement component C3, and S. mutans antigens in the glomeruli. Immunoglobulin G eluted from nephritic kidneys reacted in immunoblots with the 24,000, 35,000, and 65,000 Mr components of S. mutans extract, indicating that the antigens that bound to tissue in vitro also bound in vivo and reacted with antibodies in situ. Antibodies to other S. mutans antigens were not detected in the kidney eluate, although they were present in the serum of the same rabbit. Images PMID:6384042

  9. Two outer membrane proteins are bovine lactoferrin-binding proteins in Mannheimia haemolytica A1.

    PubMed

    Samaniego-Barrón, Luisa; Luna-Castro, Sarahí; Piña-Vázquez, Carolina; Suárez-Güemes, Francisco; de la Garza, Mireya

    2016-01-01

    Mannheimia haemolytica is a Gram negative bacterium that is part of the bovine respiratory disease, which causes important economic losses in the livestock industry. In the present work, the interaction between M. haemolytica A1 and bovine lactoferrin (BLf) was studied. This iron-chelating glycoprotein is part of the mammalian innate-immune system and is present in milk and mucosal secretions; Lf is also contained in neutrophils secondary granules, which release this glycoprotein at infection sites. It was evidenced that M. haemolytica was not able to use iron-charged BLf (BholoLf) as a sole iron source; nevertheless, iron-lacked BLf (BapoLf) showed a bactericidal effect against M. haemolytica with MIC of 4.88 ± 1.88 and 7.31 ± 1.62 μM for M. haemolytica strain F (field isolate) and M. haemolytica strain R (reference strain), respectively. Through overlay assays and 2-D electrophoresis, two OMP of 32.9 and 34.2 kDa with estimated IP of 8.18 and 9.35, respectively, were observed to bind both BapoLf and BholoLf; these OMP were identified by Maldi-Tof as OmpA (heat-modifiable OMP) and a membrane protein (porin). These M. haemolytica BLf binding proteins could be interacting in vivo with both forms of BLf depending on the iron state of the bovine. PMID:27599994

  10. A New Type I Peritrophic Membrane Protein from Larval Holotrichia oblita (Coleoptera: Melolonthidae) Binds to Chitin

    PubMed Central

    Liu, Xiaomin; Li, Jie; Guo, Wei; Li, Ruijun; Zhao, Dan; Li, Xinna

    2014-01-01

    Peritrophic membranes (PMs) are composed of chitin and protein. Chitin and protein play important roles in the structural formation and function of the PM. A new type I PM protein, HoCBP76, was identified from the Holotrichia oblita. HoCBP76 was shown as a 62.3 kDa protein by SDS-PAGE analysis and appeard to be associated with the PM throughout its entire length. In H. oblita larvae, the midgut is the only tissue where HoCBP76 could be detected during the feeding period of the larvae. The predicted amino acid sequence indicates that it contains seven tandem chitin binding domains belonging to the peritrophin-A family. HoCBP76 has chitin binding activity and is strongly associated with the PM. The HoCBP76 was not a mucin-like glycoprotein, and the consensus of conserved cysteines appeared to be CX13–17CX5CX9CX12CX7C. Western blot analysis showed that the abundance of HoCBP76 in the anterior, middle and posterior regions of the midgut was similar, indicating that HoCBP76 was secreted by the whole midgut epithelium, and confirmed the H. oblita PM belonged to the Type I PM. Immunolocalization analysis showed that HoCBP76 was mainly localized in the PM. The HoCBP76 is the first PM protein found in the H. oblita; however, its biochemical and physiological functions require further investigation. PMID:24758927

  11. Identification of a new binding protein for crotoxin and other neurotoxic phospholipase A sub 2 s on brain synaptic membranes

    SciTech Connect

    Chonho Yen ); Muchin Tzeng Academia Sinica, Taipei )

    1991-12-03

    Crotoxin and other neurotoxic phospholipase A{sub 2}s exert neurotoxicity by acting primarily at the presynaptic level. Strong binding of crotoxin and several other to synaptic membranes has been demonstrated previously. In this study the authors used simple chemical cross-linking techniques to identify the neuronal membrane molecules involved in the binding of these toxins. After {sup 125}I-crotoxin had bound to synaptosomes from guinea pig brain, treatment with disuccinimidyl suberate, disuccinimidyl dithiobis(propionate) or ethylene glycol bis(succinimidyl succinate) resulted in the formation of a predominant radioactive conjugate of {approximately}60 kDa, which was different from the conjugate formed by photoaffinity labeling technique in a previous report. The membrane component in the conjugate was shown to be a single-chain protein {approximately}45 kDa. In subfractions of synaptosomes, this binding protein was mostly found in the synaptic membrane fraction and was not present in the mitochondrial fraction. Plasma membranes from several nonneural tissues also did not contain this binding protein. Unmodified crotoxin inhibited the formation o this adduct with an IC{sub 50} of around 1 {times} 10{sup {minus}8} M. Mojave toxin and some other phospholipase A{sub 2}s were also highly inhibitory to this conjugation, and notexin and others were less effective, while {beta}-bungarotoxin and pancreatic PLA{sub 2} were totally ineffective. They concluded that a new protein of 45 kDa specifically present in neuronal membranes is another major molecule responsible for the binding of crotoxin and other phospholipase A{sub 2}s.

  12. Ca2+ and membrane binding to annexin 3 modulate the structure and dynamics of its N terminus and domain III

    PubMed Central

    Sopkova, Jana; Raguenes-Nicol, Céline; Vincent, Michel; Chevalier, Anne; Lewit-Bentley, Anita; Russo-Marie, Françoise; Gallay, Jacques

    2002-01-01

    Annexin 3 (ANX A3) represents ∼1% of the total protein of human neutrophils and promotes tight contact between membranes of isolated specific granules in vitro leading to their aggregation. Like for other annexins, the primary molecular events of the action of this protein is likely its binding to negatively charged phospholipid membranes in a Ca2+-dependent manner, via Ca2+-binding sites located on the convex side of the highly conserved core of the molecule. The conformation and dynamics of domain III can be affected by this process, as it was shown for other members of the family. The 20 amino-acid, N-terminal segment of the protein also could be affected and also might play a role in the modulation of its binding to the membranes. The structure and dynamics of these two regions were investigated by fluorescence of the two tryptophan residues of the protein (respectively, W190 in domain III and W5 in the N-terminal segment) in the wild type and in single-tryptophan mutants. By contrast to ANX A5, which shows a closed conformation and a buried W187 residue in the absence of Ca2+, domain III of ANX A3 exhibits an open conformation and a widely solvent-accessible W190 residue in the same conditions. This is in agreement with the three-dimensional structure of the ANX A3-E231A mutant lacking the bidentate Ca2+ ligand in domain III. Ca2+ in the millimolar concentration range provokes nevertheless a large mobility increase of the W190 residue, while interaction with the membranes reduces it slightly. In the N-terminal region, the W5 residue, inserted in the central pore of the protein, is weakly accessible to the solvent and less mobile than W190. Its amplitude of rotation increases upon binding of Ca2+ and returns to its original value when interacting with membranes. Ca2+ concentration for half binding of the W5A mutant to negatively charged membranes is ∼0.5 mM while it increases to ∼1 mM for the ANX A3 wild type and to ∼3 mM for the W190 ANX A3 mutant. In

  13. Identification of leukotriene D4 specific binding sites in the membrane preparation isolated from guinea pig lung

    SciTech Connect

    Mong, S.; Wu, H.L.; Clark, M.A.; Stadel, J.M.; Gleason, J.G.; Crooke, S.T.

    1984-12-01

    A radioligand binding assay has been established to study leukotriene specific binding sites in the guinea pig and rabbit tissues. Using high specific activity (/sup 3/H)-leukotriene D4 (( /sup 3/H)-LTD4), in the presence or absence of unlabeled LTD4, the diastereoisomer of LTD4 (5R,6S-LTD4), leukotriene E4 (LTE4) and the end-organ antagonist, FPL 55712, the authors have identified specific binding sites for (/sup 3/H)-LTD4 in the crude membrane fraction isolated from guinea pig lung. The time required for (/sup 3/H)-LTD4 binding to reach equilibrium was approximately 20 to 25 min at 37 degrees C in the presence of 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The binding of (/sup 3/H)-LTD4 to the specific sites was saturable, reversible and stereospecific. The maximal number of binding sites (Bmax), derived from Scatchard analysis, was approximately 320 +/- 200 fmol per mg of crude membrane protein. The dissociation constants, derived from kinetic and saturation analyses, were 9.7 nM and 5 +/- 4 nM, respectively. The specific binding sites could not be detected in the crude membrane fraction prepared from guinea pig ileum, brain and liver, or rabbit lung, trachea, ileum and uterus. In radioligand competition experiments, LTD4, FPL 55712 and 5R,6S-LTD4 competed with (/sup 3/H)-LTD4. The metabolic inhibitors of arachidonic acid and SKF 88046, an antagonist of the indirectly-mediated actions of LTD4, did not significantly compete with (/sup 3/H)-LTD4 at the specific binding sites. These correlations indicated that these specific binding sites may be the putative leukotriene receptors in the guinea-pig lung.

  14. Role of the Fast Kinetics of Pyroglutamate-Modified Amyloid-β Oligomers in Membrane Binding and Membrane Permeability

    PubMed Central

    2015-01-01

    Membrane permeability to ions and small molecules is believed to be a critical step in the pathology of Alzheimer’s disease (AD). Interactions of oligomers formed by amyloid-β (Aβ) peptides with the plasma cell membrane are believed to play a fundamental role in the processes leading to membrane permeability. Among the family of Aβs, pyroglutamate (pE)-modified Aβ peptides constitute the most abundant oligomeric species in the brains of AD patients. Although membrane permeability mechanisms have been studied for full-length Aβ1–40/42 peptides, these have not been sufficiently characterized for the more abundant AβpE3–42 fragment. Here we have compared the adsorbed and membrane-inserted oligomeric species of AβpE3–42 and Aβ1–42 peptides. We find lower concentrations and larger dimensions for both species of membrane-associated AβpE3–42 oligomers. The larger dimensions are attributed to the faster self-assembly kinetics of AβpE3–42, and the lower concentrations are attributed to weaker interactions with zwitterionic lipid headgroups. While adsorbed oligomers produced little or no significant membrane structural damage, increased membrane permeabilization to ionic species is understood in terms of enlarged membrane-inserted oligomers. Membrane-inserted AβpE3–42 oligomers were also found to modify the mechanical properties of the membrane. Taken together, our results suggest that membrane-inserted oligomers are the primary species responsible for membrane permeability. PMID:24950761

  15. Protein-binding site at the immunoglobulin mu membrane polyadenylylation signal: possible role in transcription termination.

    PubMed Central

    Law, R; Kuwabara, M D; Briskin, M; Fasel, N; Hermanson, G; Sigman, D S; Wall, R

    1987-01-01

    mRNAs specifying immunoglobulin mu and delta heavy chains are encoded by a single large, complex transcription unit (mu + delta gene). The transcriptional activity of delta gene segments in terminally differentiated, IgM-secreting B lymphocytes is 10-20 times lower than in earlier B-lineage cells expressing delta mRNA. We find that transcription of the mu + delta gene in IgM-secreting murine myeloma cells terminates within a region of 500-1000 nucleotides immediately following the mu membrane (mu m) polyadenylylation site. Transcription decreases only minimally through this region in murine cell lines representative of earlier stages in B-cell development. A DNA fragment containing the mu m polyadenylylation signal gives protein-DNA complexes with different mobilities in gel retardation assays with nuclear extracts from myeloma cells than with nuclear extracts from earlier B-lineage cells. However, using a recently developed "footprinting" procedure in which protein-DNA complexes resolved in gel retardation assays are subjected to nucleolytic cleavage while still in the polyacrylamide gel, we find that the DNA sequences protected by factors from the two cell types are indistinguishable. The factor-binding site on the DNA is located 5' of the mu m polyadenylylation signal AATAAA and includes the 15-nucleotide-long A + T-rich palindrome CTGTAAACAAATGTC. This type of palindromic binding site exhibits orientation-dependent activity consistent with the reported properties of polymerase II termination signals. This binding site is followed by two sets of directly repeated DNA sequences with different helical conformation as revealed by their reactivity with the chemical nuclease 1,10-phenanthroline-copper. The close proximity of these features to the signals for mu m mRNA processing may reflect a linkage of the processes of developmentally regulated mu m polyadenylylation and transcription termination. Images PMID:3122214

  16. Structural basis for androgen specificity and oestrogen synthesis in human aromatase

    SciTech Connect

    Ghosh, Debashis; Griswold, Jennifer; Erman, Mary; Pangborn, Walter

    2009-03-06

    Aromatase cytochrome P450 is the only enzyme in vertebrates known to catalyse the biosynthesis of all oestrogens from androgens. Aromatase inhibitors therefore constitute a frontline therapy for oestrogen-dependent breast cancer. In a three-step process, each step requiring 1 mol of O{sub 2}, 1 mol of NADPH, and coupling with its redox partner cytochrome P450 reductase, aromatase converts androstenedione, testosterone and 16{alpha}-hydroxytestosterone to oestrone, 17{beta}-oestradiol and 17{beta},16{alpha}-oestriol, respectively. The first two steps are C19-methyl hydroxylation steps, and the third involves the aromatization of the steroid A-ring, unique to aromatase. Whereas most P450s are not highly substrate selective, it is the hallmark androgenic specificity that sets aromatase apart. The structure of this enzyme of the endoplasmic reticulum membrane has remained unknown for decades, hindering elucidation of the biochemical mechanism. Here we present the crystal structure of human placental aromatase, the only natural mammalian, full-length P450 and P450 in hormone biosynthetic pathways to be crystallized so far. Unlike the active sites of many microsomal P450s that metabolize drugs and xenobiotics, aromatase has an androgen-specific cleft that binds the androstenedione molecule snugly. Hydrophobic and polar residues exquisitely complement the steroid backbone. The locations of catalytically important residues shed light on the reaction mechanism. The relative juxtaposition of the hydrophobic amino-terminal region and the opening to the catalytic cleft shows why membrane anchoring is necessary for the lipophilic substrates to gain access to the active site. The molecular basis for the enzyme's androgenic specificity and unique catalytic mechanism can be used for developing next-generation aromatase inhibitors.

  17. Specific binding of a ligand of sigma-opioid receptors - N-allylnormetazocine (SKF 10047) - with liver membranes

    SciTech Connect

    Samovilova, N.N.; Yarygin, K.N.; Vinogradov, V.A.

    1986-08-01

    A ligand of the sigma-opioid receptors - N-allylnormetazocine (SKF 10047) -binds specifically and reversible with rat liver membranes. In relation to a number of properties, the sites binding SKF 10047 in the liver are similar to the sigma-opioid receptors of the central nervous system. They do not interact with classical opiates (morphine, naloxone) and with opioid peptides, but bind well benzomorphans (bremazocine, SKF 10047) and a number of compounds of different chemical structures with a pronounced psychtropic action (haloperidol, imipramine, phencyclidine, etc.).

  18. Relationships between Membrane Binding, Affinity and Cell Internalization Efficacy of a Cell-Penetrating Peptide: Penetratin as a Case Study

    PubMed Central

    Alves, Isabel D.; Bechara, Cherine; Walrant, Astrid; Zaltsman, Yefim; Jiao, Chen-Yu; Sagan, Sandrine

    2011-01-01

    Background Penetratin is a positively charged cell-penetrating peptide (CPP) that has the ability to bind negatively charged membrane components, such as glycosaminoglycans and anionic lipids. Whether this primary interaction of penetratin with these cell surface components implies that the peptide will be further internalized is not clear. Methodology Using mass spectrometry, the amount of internalized and membrane bound penetratin remaining after washings, were quantified in three different cell lines: wild type (WT), glycosaminoglycans- (GAGneg) and sialic acid-deficient (SAneg) cells. Additionally, the affinity and kinetics of the interaction of penetratin to membrane models composed of pure lipids and membrane fragments from the referred cell lines was investigated, as well as the thermodynamics of such interactions using plasmon resonance and calorimetry. Principal Findings Penetratin internalized with the same efficacy in the three cell lines at 1 µM, but was better internalized at 10 µM in SAneg>WT>GAGneg. The heat released by the interaction of penetratin with these cells followed the ranking order of internalization efficiency. Penetratin had an affinity of 10 nM for WT cells and µM for SAneg and GAGneg cells and model membrane of phospholipids. The remaining membrane-bound penetratin after cells washings was similar in WT and GAGneg cells, which suggested that these binding sites relied on membrane phospholipids. The interaction of penetratin with carbohydrates was more superficial and reversible while it was stronger with phospholipids, likely because the peptide can intercalate between the fatty acid chains. Conclusion/Significance These results show that accumulation and high-affinity binding of penetratin at the cell-surface do not reflect the internalization efficacy of the peptide. Altogether, these data further support translocation (membrane phospholipids interaction) as being the internalization pathway used by penetratin at low

  19. Structural and biochemical studies reveal UbiG/Coq3 as a class of novel membrane-binding proteins.

    PubMed

    Zhu, Yuwei; Wu, Bo; Zhang, Xu; Fan, Xiaojiao; Niu, Liwen; Li, Xu; Wang, Junfeng; Teng, Maikun

    2015-08-15

    UbiG and Coq3 (orthologue in eukaryotes) are SAM-MTases (S-adenosylmethionine-dependent methyltransferases) that catalyse both O-methylation steps in CoQ biosynthesis from prokaryotes to eukaryotes. However, the detailed molecular mechanism by which they function remains elusive. In the present paper, we report that UbiG/Coq3 defines a novel class of membrane-binding proteins. Escherichia coli UbiG binds specifically to liposomes containing PG (phosphatidylglycerol) or CL (cardiolipin, or diphosphatidylglycerol), two major lipid components of the E. coli plasma membrane, whereas human and yeast Coq3 display a strong preference for liposomes enriched with CL, a signature lipid of the mitochondrial membrane. The crystal structure of UbiG from E. coli was determined at 2.1 Å (1 Å = 0.1 nm) resolution. The structure exhibits a typical Class I SAM-MTase fold with several variations, including a unique insertion between strand β5 and helix α10. This insertion is highly conserved and is required for membrane binding. Mutation of the key residues renders UbiG unable to efficiently bind liposome in vitro and the mutant fails to rescue the phenotype of ΔubiG strain in vivo. Taken together, our results shed light on a novel biochemical function of the UbiG/Coq3 protein. PMID:26251450

  20. Binding of glycosaminoglycans to cyano-activated agarose membranes: kinetic and diffusional effects on yield and homogeneity.

    PubMed

    Mattern, Kristin J; Deen, William M

    2007-11-01

    Methods were developed for binding a glycosaminoglycan (GAG, a 50 kDa chondroitin sulfate) to thin agarose membranes using 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) as the activating agent. Process conditions were optimized to achieve high yields and spatially uniform concentrations of bound ligand. Yields were varied mainly by manipulating the duration and temperature of the aqueous washes prior to coupling, which affected the concentration of active sites available for subsequent GAG binding. The rate constants for degradation of the active cyanate esters in 0.1M bicarbonate solutions were 0.24+/-0.02 h(-1) at 4 degrees C and 0.08+/-0.03 h(-1) at 0 degrees C. Steric limitations in the 3% agarose gels severely restricted binding, with only about 0.1% of active sites being accessible to GAG molecules. The GAG binding occurred primarily in the outer 50-70 microm of the membranes, so that coupling was homogeneous only for thin gels. A model of GAG diffusion and reaction in the coupling step was developed to explain the observed effects of parameters such as the GAG concentration in solution and the membrane thickness. An analysis of the key time scales in the synthesis provides design principles that should be useful also for other cyanylating agents, other ligands, and for beads as well as membranes. PMID:17610855

  1. Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

    SciTech Connect

    Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.; Wang, Yu-Hsiu; Slochower, David; Janmey, Paul A.; Lemmon, Mark A.

    2011-09-28

    Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importance of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.

  2. Rapid effects of androgens in macrophages.

    PubMed

    Benten, W Peter M; Guo, Z; Krücken, J; Wunderlich, F

    2004-08-01

    We investigated the existence of membrane receptors for testosterone (mAR) in mouse macrophages of the cell lines IC-21 and RAW 264.7 as well as their roles in nongenomic pathways, gene expression and cell functioning. Both cell lines lack intracellular androgen receptors (iARs) and respond to testosterone with rapid rises in [Ca2+]i. These rises in [Ca2+]i can neither be inhibited by iAR- nor by iER blockers, but are rather mediated through mAR. Pharmacological approaches suggest that the mAR belongs to the class of membrane receptors which are coupled to phospholipase C via pertussis toxin (PTX) sensitive G-proteins. The mAR can be localized as specific surface binding sites for testosterone-BSA-FITC by confocal laser scanning microscopy (CLSM)and flow cytometry, and are characterized by their agonist-sequestrability. In order to examine a possible role of the testosterone-induced rise in [Ca2+]i on gene expression, a c-fos promoter reporter gene construct was transfected into RAW 264.7 macrophages. The increase in [Ca2+]i induced by testosterone cannot significantly activate the c-fos promoter directly. Also, no significant activation of ERK1/2, JNK/SAPK and p38 can be observed following testosterone-stimulation alone. However, testosterone-induced rises in [Ca2+]i do have specific effects on gene expression in context with lipopolysaccharide (LPS)-induced genotropic signaling: testosterone specifically down-regulates LPS-induced activation of c-fos promoter, p38 MAPK and NO production. In fetal calf serum (FCS)-induced genotropic signaling, the situation is reversed, i.e. testosterone augments the activation of c-fos promoter and ERK1/2. Our studies demonstrate a cross-talk between the testosterone-induced nongenomic Ca2+ signaling and the genotropic signaling induced by LPS and FCS in macrophages. PMID:15288774

  3. Two successive calcium-dependent transitions mediate membrane binding and oligomerization of daptomycin and the related antibiotic A54145.

    PubMed

    Taylor, Robert; Butt, Khalida; Scott, Bradley; Zhang, TianHua; Muraih, Jawad K; Mintzer, Evan; Taylor, Scott; Palmer, Michael

    2016-09-01

    Daptomycin and A54145 are homologous lipopeptide antibiotics that permeabilize the cell membranes of Gram-positive bacteria. Membrane permeabilization depends on the presence of both phosphatidylglycerol (PG) and calcium, and it involves the formation of oligomeric transmembrane pores that consist of approximately 6-8 subunits. We here show that each lipopeptide molecule binds two calcium ions in separable, successive steps. The first calcium ion causes the lipopeptide molecule to bind to the target membrane, and likely to form a loosely associated oligomer. Higher calcium concentrations induce binding of a second ion, which produces the more tightly associated and more deeply membrane-inserted final, functional form of the oligomer. Both calcium-dependent steps are accompanied by fluorescence signals that indicate transition of specific amino acid residues into less polar environments, suggestive of insertion into the target membrane. Our findings agree with the earlier observation that two of the four acidic amino acid residues in the daptomycin molecule are essential for antibacterial activity. PMID:27237728

  4. Probing conformational changes in lipoxygenases upon membrane binding: fine-tuning by the active site inhibitor ETYA.

    PubMed

    Di Venere, Almerinda; Nicolai, Eleonora; Ivanov, Igor; Dainese, Enrico; Adel, Susan; Angelucci, B C; Kuhn, Hartmut; Maccarrone, Mauro; Mei, Giampiero

    2014-01-01

    Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that are involved in the metabolism of polyunsaturated fatty acids. Their biological activity includes a membrane binding process whose molecular details are not completely understood. The mechanism of enzyme-membrane interactions is thought to involve conformational changes at the level of the protein tertiary structure, and the extent of such alterations depends on the degree of structural flexibility of the different LOX isoforms. In this study, we have tested the resilience properties of a plant and a mammalian LOX, by using high pressure fluorescence measurements at different temperatures. The binding of LOXs to the lipid bilayer has been characterized using both large and giant unilamellar vesicles and electron transfer particles (inner mitochondrial membranes) as model membranes. The data indicate that the degree of LOXs' flexibility is strictly dependent on the two distinct N- and C-terminal domains that characterize the 3D structure of these enzymes. Furthermore, they demonstrate that increasing the rigidity of protein scaffolding by the presence of an active site ligand impairs the membrane binding ability of LOXs. These findings provide evidence that the amphitropic nature of LOXs is finely tuned by the interaction of the substrate with the residues of the active site, suggesting new strategies for the design of enzyme inhibitors. PMID:24012824

  5. Local changes in lipid environment of TCR microclusters regulate membrane binding by the CD3ε cytoplasmic domain

    PubMed Central

    Schubert, David A.; Gordo, Susana; Chu, H. Hamlet

    2012-01-01

    The CD3ε and ζ cytoplasmic domains of the T cell receptor bind to the inner leaflet of the plasma membrane (PM), and a previous nuclear magnetic resonance structure showed that both tyrosines of the CD3ε immunoreceptor tyrosine-based activation motif partition into the bilayer. Electrostatic interactions between acidic phospholipids and clusters of basic CD3ε residues were previously shown to be essential for CD3ε and ζ membrane binding. Phosphatidylserine (PS) is the most abundant negatively charged lipid on the inner leaflet of the PM and makes a major contribution to membrane binding by the CD3ε cytoplasmic domain. Here, we show that TCR triggering by peptide–MHC complexes induces dissociation of the CD3ε cytoplasmic domain from the plasma membrane. Release of the CD3ε cytoplasmic domain from the membrane is accompanied by a substantial focal reduction in negative charge and available PS in TCR microclusters. These changes in the lipid composition of TCR microclusters even occur when TCR signaling is blocked with a Src kinase inhibitor. Local changes in the lipid composition of TCR microclusters thus render the CD3ε cytoplasmic domain accessible during early stages of T cell activation. PMID:23166358

  6. Artificial masculinization in tilapia involves androgen receptor activation.

    PubMed

    Golan, Matan; Levavi-Sivan, Berta

    2014-10-01

    Estrogens have a pivotal role in natural female sexual differentiation of tilapia while lack of steroids results in testicular development. Despite the fact that androgens do not participate in natural sex differentiation, synthetic androgens, mainly 17-α-methyltestosterone (MT) are effective in the production of all-male fish in aquaculture. The sex inversion potency of synthetic androgens may arise from their androgenic activity or else as inhibitors of aromatase activity. The current study is an attempt to differentiate between the two alleged activities in order to evaluate their contribution to the sex inversion process and aid the search for novel sex inversion agents. In the present study, MT inhibited aromatase activity, when applied in vitro as did the non-aromatizable androgen dihydrotestosterone (DHT). In comparison, exposure to fadrozole, a specific aromatase inhibitor, was considerably more effective. Androgenic activity of MT was evaluated by exposure of Sciaenochromis fryeri fry to the substance and testing for the appearance of blue color. Flutamide, an androgen antagonist, administered concomitantly with MT, reduced the appearance of the blue color and the sex inversion potency of MT in a dose-dependent manner. In tilapia, administration of MT, fadrozole or DHT resulted in efficient sex inversion while flutamide reduced the sex inversion potency of all three compounds. In the case of MT and DHT the decrease in sex inversion efficiency caused by flutamide is most likely due to the direct blocking of the androgen binding to its cognate receptor. The negative effect of flutamide on the efficiency of the fadrozole treatment may indicate that the masculinizing activity of fadrozole may be attributed to excess, un-aromatized, androgens accumulated in the differentiating gonad. The present study shows that when androgen receptors are blocked, there is a reduction in the efficiency of sex inversion treatments. Our results suggest that in contrast to

  7. Androgens and prostate disease

    PubMed Central

    Cooper, Lori A; Page, Stephanie T

    2014-01-01

    A growing body of literature has established the anabolic benefits of testosterone (T) therapy in hypogonadal men. However, there remains a paucity of data regarding the risks of exogenous androgen use in older men and the potential for adverse effects on the prostate gland. Whether T therapy in older, hypogonadal men might worsen lower urinary tract symptoms or exacerbate, unmask, or even incite prostate cancer development has tempered enthusiasm for T therapy, while known prostatic disease has served as a relative contraindication to T therapy. Androgens are necessary for the development and maintenance of the prostate gland. However, epidemiologic studies do not consistently find a positive relationship between endogenous serum androgen concentrations and the risk of prostate disease. Recent data demonstrate that 5α-reductase inhibitors decrease the risk of low-grade prostate cancer, suggesting that modifying androgen metabolism may have beneficial effects on prostate health, yet similar reductions in high-grade disease have not been observed, thereby questioning the true clinical benefits of these agents for chemoprevention. Knowing how to best investigate the relationship between androgens and the development of prostate disease given the lack of large, randomized trials is difficult. Accumulating data challenges the assumption that alterations in serum androgens have parallel effects within the prostate hormonal environment or change androgen-regulated processes within the gland. Long-term intervention studies are needed to truly ascertain the effects of androgen manipulation on prostate tissue and disease risk. However, available data do not support the notion that restoring serum androgens to normal physiologic ranges drives prostate disease. PMID:24407178

  8. In vitro screening of psychoactive drugs by [(35)S]GTPgammaS binding in rat brain membranes.

    PubMed

    Nonaka, Ryouichi; Nagai, Fumiko; Ogata, Akio; Satoh, Kanako

    2007-12-01

    We constructed a reproducible, simple, and small-scale determination method of the psychoactive drugs that acted directly on the monoamine receptor by measuring the activation of [(35)S]guanosine-5'-O-(3-thio)-triphosphate binding to guanine nucleotide-binding proteins (G proteins). This method can simultaneously measure the effects of three monoamines, namely dopamine (DA), serotonin (5-HT), and norepinephrine (NE), in rat brain membranes using a 96-well microplate. Activation of D(1) and D(2) receptors in striatal membranes by DA as well as 5-HT and NEalpha(2) receptors in cortical membranes could be measured. Of 12 tested phenethylamines, 2,5-dimethoxy-4-chlorophenethylamine (2C-C), 2,5-dimethoxy-4-ethylphenethylamine (2C-E), and 2,5-dimethoxy-4-iodophenethylamine (2C-I) stimulated G protein binding. The other phenethylamines did not affect G protein binding. All 7 tryptamines tested stimulated G protein binding with the following rank order of potency; 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT)>5-methoxy-N,N-diallyltryptamine (5-MeO-DALT)>5-methoxy-alpha-methyltryptamine (5-MeO-AMT)>or=5-methoxy-N,N-methylisopropyltryptamine (5-MeO-MIPT)>5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT)>N,N-dipropyltryptamine (DPT)>or=alpha-methyltryptamine (AMT). This assay system was able to designate psychoactive drugs as prohibited substances in accordance with criteria set forth by the Tokyo Metropolitan government. PMID:18057721

  9. Determination of mercury in river water by diffusive gradients in thin films using P81 membrane as binding layer.

    PubMed

    Colaço, Camila Destro; Yabuki, Lauren Nozomi Marques; Rolisola, Ana Marta; Menegário, Amauri Antonio; de Almeida, Eduardo; Suárez, Carlos Alfredo; Gao, Yue; Corns, Warren T; do Nascimento Filho, Virgílio Franco

    2014-11-01

    In this work, a device based on diffusive gradients in thin films (DGT) was evaluated for the determination of Hg(II) in river water. The DGT device was assembled with a cellulose phosphate ion exchange membrane (P81 Whatman) as a binding phase and agarose gel 1.5% (m/v) as a diffusive layer. Laboratory deployments showed that the binding of Hg(2+) ([Hg(DGT)]/[Hg(solution)]) by P81 membrane was more effective (97%) than the Chelex 100 resin (80%).The effect of ionic strength, pH and potential interfering ions on Hg binding with DGT׳s was investigated. The results showed no significant effect on the binding of Hg(II) at pH range from 3.5 to 8.5 and at an ionic strength range from 0.0005 to 0.1 mol L(-1). Uptakes of 50 µg L(-1) Hg(II) by P81 membrane were not affected by Fe, Mn, Zn, Cu, Ca and Mg at the concentration range of 200-1800 µg L(-1). Finally, the DGT device using the P81 as the binding layer was applied for in situ measurements of Hg in river water. For in situ measurements, the labile Hg concentration (from <2 to 13 ng L(-1)) was lower than 10% of the dissolved fraction (from 155 to 446 ng L(-1)). PMID:25127614

  10. The effects of down-regulating expression of Arabidopsis thaliana membrane-associated acyl-CoA binding protein 2 on acyl-lipid composition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multiple classes of acyl-CoA binding proteins are encoded by plant genomes, including a plant-unique class of predicted integral membrane-proteins. Transcript analysis revealed that both of the integral membrane-acyl-CoA binding proteins of Arabidopsis thaliana, ACBP1 and ACBP2, are expressed in al...

  11. Lectin-binding sites and silver affinity of the macula densa basement membranes in the rabbit kidney.

    PubMed Central

    Ojeda, J L; Piedra, S

    1994-01-01

    Fluorochrome-labelled lectins and the Jones method of silver impregnation preceded by different oxidation and enzymatic digestion procedures were used to study the patterns of glycosylation and silver affinity of the macula densa (MD) and thick ascending limb (TAL) basement membranes of the rabbit kidney. The goal of this study was to analyse the morphological basis of MD basement membrane permeability and its possible role in modulation of the signal involved in tubuloglomerular feedback control of the juxtaglomerular apparatus. The lectin-binding pattern and silver affinity of basement membrane differed clearly from those of the TAL basement membrane. The former had greater WGA and Con A affinity than the latter. Furthermore, the MD basement membrane lost argyrophilia in permanganate oxidized sections whereas that of the TAL did not. The cell coat of MD cells differed from that of the TAL cells in that it had N-acetyl neuraminic acid and Con A binding sites. Our results suggest that the MD basement membrane has a distinctive macromolecular composition which may be related to its permeability to high molecular weight molecules. Images Fig. 1 Fig. 2 Fig. 3 PMID:7544331

  12. Testosterone Reduces Knee Passive Range of Motion and Expression of Relaxin Receptor Isoforms via 5α-Dihydrotestosterone and Androgen Receptor Binding

    PubMed Central

    Dehghan, Firouzeh; Muniandy, Sekaran; Yusof, Ashril; Salleh, Naguib

    2014-01-01

    Ovarian steroids such as estrogen and progesterone have been reported to influence knee laxity. The effect of testosterone, however, remains unknown. This study investigated the effect of testosterone on the knee range of motion (ROM) and the molecular mechanisms that might involve changes in the expression of relaxin receptor isoforms, Rxfp1 and Rxfp2 in the patella tendon and lateral collateral ligament of the female rat knee. Ovariectomized adult female Wistar rats received three days treatment with peanut oil (control), testosterone (125 and 250 μg/kg) and testosterone (125 and 250 μg/kg) plus flutamide, an androgen receptor blocker or finasteride, a 5α-reductase inhibitor. Duplicate groups received similar treatment however in the presence of relaxin (25 ng/kg). A day after the last drug injection, knee passive ROM was measured by using a digital miniature goniometer. Both tendon and ligament were harvested and then analysed for protein and mRNA expression for Rxfp1 and Rxfp2 respectively. Knee passive ROM, Rxfp1 and Rxfp2 expression were significantly reduced following treatment with testosterone. Flutamide or finasteride administration antagonized the testosterone effect. Concomitant administration of testosterone and relaxin did not result in a significant change in knee ROM as compared to testosterone only treatment; however this was significantly increased following flutamide or finasteride addition. Testosterone effect on knee passive ROM is likely mediated via dihydro-testosterone (DHT), and involves downregulation of Rxfp1 and Rxfp2 expression, which may provide the mechanism underlying testosterone-induced decrease in female knee laxity. PMID:24642882

  13. Testosterone reduces knee passive range of motion and expression of relaxin receptor isoforms via 5α-dihydrotestosterone and androgen receptor binding.

    PubMed

    Dehghan, Firouzeh; Muniandy, Sekaran; Yusof, Ashril; Salleh, Naguib

    2014-01-01

    Ovarian steroids such as estrogen and progesterone have been reported to influence knee laxity. The effect of testosterone, however, remains unknown. This study investigated the effect of testosterone on the knee range of motion (ROM) and the molecular mechanisms that might involve changes in the expression of relaxin receptor isoforms, Rxfp1 and Rxfp2 in the patella tendon and lateral collateral ligament of the female rat knee. Ovariectomized adult female Wistar rats received three days treatment with peanut oil (control), testosterone (125 and 250 μg/kg) and testosterone (125 and 250 μg/kg) plus flutamide, an androgen receptor blocker or finasteride, a 5α-reductase inhibitor. Duplicate groups received similar treatment however in the presence of relaxin (25 ng/kg). A day after the last drug injection, knee passive ROM was measured by using a digital miniature goniometer. Both tendon and ligament were harvested and then analysed for protein and mRNA expression for Rxfp1 and Rxfp2 respectively. Knee passive ROM, Rxfp1 and Rxfp2 expression were significantly reduced following treatment with testosterone. Flutamide or finasteride administration antagonized the testosterone effect. Concomitant administration of testosterone and relaxin did not result in a significant change in knee ROM as compared to testosterone only treatment; however this was significantly increased following flutamide or finasteride addition. Testosterone effect on knee passive ROM is likely mediated via dihydro-testosterone (DHT), and involves downregulation of Rxfp1 and Rxfp2 expression, which may provide the mechanism underlying testosterone-induced decrease in female knee laxity. PMID:24642882

  14. Interactions between Plasmodium falciparum skeleton-binding protein 1 and the membrane skeleton of malaria-infected red blood cells

    PubMed Central

    Buckingham, Donna W.; Blanc, Lionel; Hale, John; Guo, Xinhua; Pei, Xinhong; Herrmann, Susann; Hanssen, Eric G.; Coppel, Ross L.; Mohandas, Narla; An, Xiuli; Cooke, Brian M.

    2015-01-01

    During development inside red blood cells (RBCs), Plasmodium falciparum malaria parasites export proteins that associate with the RBC membrane skeleton. These interactions cause profound changes to the biophysical properties of RBCs that underpin the often severe and fatal clinical manifestations of falciparum malaria. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is one such exported parasite protein that plays a major role in malaria pathogenesis since its exposure on the parasitised RBC surface mediates their adhesion to vascular endothelium and placental syncytioblasts. En route to the RBC membrane skeleton, PfEMP1 transiently associates with Maurer's clefts (MCs), parasite-derived membranous structures in the RBC cytoplasm. We have previously shown that a resident MC protein, skeleton-binding protein 1 (SBP1), is essential for the placement of PfEMP1 onto the RBC surface and hypothesised that the function of SBP1 may be to target MCs to the RBC membrane. Since this would require additional protein interactions, we set out to identify binding partners for SBP1. Using a combination of approaches, we have defined the region of SBP1 that binds specifically to defined subdomains of two major components of the RBC membrane skeleton, protein 4.1R and spectrin. We show that these interactions serve as one mechanism to anchor MCs to the RBC membrane skeleton, however, while they appear to be necessary, they are not sufficient for the translocation of PfEMP1 onto the RBC surface. The N-terminal domain of SBP1 that resides within the lumen of MCs clearly plays an essential, but presently unknown role in this process. PMID:25883090

  15. Targeting and insertion of the cholesterol-binding translocator protein into the outer mitochondrial membrane.

    PubMed

    Rone, Malena B; Liu, Jun; Blonder, Josip; Ye, Xiaoying; Veenstra, Timothy D; Young, Jason C; Papadopoulos, Vassilios

    2009-07-28

    Translocator protein (18 kDa, TSPO), previously known as the peripheral-type benzodiazepine receptor, is an outer mitochondrial membrane (OMM) protein necessary for cholesterol import and steroid production. We reconstituted the mitochondrial targeting and insertion of TSPO into the OMM to analyze the signals and mechanisms required for this process. Initial studies indicated the formation of a mitochondrial 66 kDa complex through Blue Native-PAGE analysis. The formation of this complex was found to be dependent on the presence of ATP and the cytosolic chaperone Hsp90. Through mutational analysis we identified two areas necessary for TSPO targeting, import, and function: amino acids 103-108 (Schellman motif), which provide the necessary structural orientation for import, and the cholesterol-binding C-terminus required for insertion. Although the translocase of the outer mitochondrial membrane (TOM) complex proteins Tom22 and Tom40 were present in the OMM, the TOM complex did not interact with TSPO. In search of proteins involved in TSPO import, we analyzed complexes known to interact with TSPO by mass spectrometry. Formation of the 66 kDa complex was found to be dependent on an identified protein, Metaxin 1, for formation and TSPO import. The level of import of TSPO into steroidogenic cell mitochondria was increased following treatment of the cells with cAMP. These findings suggest that the initial targeting of TSPO to mitochondria is dependent upon the presence of cytosolic chaperones interacting with the import receptor Tom70. The C-terminus plays an important role in targeting TSPO to mitochondria, whereas its import into the OMM is dependent upon the presence of the Schellman motif. Final integration of TSPO into the OMM occurs via its interaction with Metaxin 1. Import of TSPO into steroidogenic cell mitochondria is regulated by cAMP. PMID:19552401

  16. The Escherichia coli Membrane Protein Insertase YidC Assists in the Biogenesis of Penicillin Binding Proteins

    PubMed Central

    de Sousa Borges, Anabela; de Keyzer, Jeanine; Driessen, Arnold J. M.

    2015-01-01

    ABSTRACT Membrane proteins need to be properly inserted and folded in the membrane in order to perform a range of activities that are essential for the survival of bacteria. The Sec translocon and the YidC insertase are responsible for the insertion of the majority of proteins into the cytoplasmic membrane. YidC can act in combination with the Sec translocon in the insertion and folding of membrane proteins. However, YidC also functions as an insertase independently of the Sec translocon for so-called YidC-only substrates. In addition, YidC can act as a foldase and promote the proper assembly of membrane protein complexes. Here, we investigate the effect of Escherichia coli YidC depletion on the assembly of penicillin binding proteins (PBPs), which are involved in cell wall synthesis. YidC depletion does not affect the total amount of the specific cell division PBP3 (FtsI) in the membrane, but the amount of active PBP3, as assessed by substrate binding, is reduced 2-fold. A similar reduction in the amount of active PBP2 was observed, while the levels of active PBP1A/1B and PBP5 were essentially similar. PBP1B and PBP3 disappeared from higher-Mw bands upon YidC depletion, indicating that YidC might play a role in PBP complex formation. Taken together, our results suggest that the foldase activity of YidC can extend to the periplasmic domains of membrane proteins. IMPORTANCE This study addresses the role of the membrane protein insertase YidC in the biogenesis of penicillin binding proteins (PBPs). PBPs are proteins containing one transmembrane segment and a large periplasmic or extracellular domain, which are involved in peptidoglycan synthesis. We observe that in the absence of YidC, two critical PBPs are not correctly folded even though the total amount of protein in the membrane is not affected. Our findings extend the function of YidC as a foldase for membrane protein (complexes) to periplasmic domains of membrane proteins. PMID:25666136

  17. Ascorbic acid prevents nonreceptor specific binding of (/sup 3/H)-5-hydroxytryptamine to bovine cerebral cortex membranes

    SciTech Connect

    Hamblin, M.W.; Adriaenssens, P.I.; Ariani, K.; Cawthon, R.M.; Stratford, C.A.; Tan, G.L.; Ciaranello, R.D.

    1987-03-01

    (/sup 3/H)-5-Hydroxytryptamine ((/sup 3/H)-5-HT) decomposes rapidly when exposed to air in solution at physiological pH if antioxidants are not present. The decomposition products appear to bind to two saturable sites on brain membranes (apparent Kd values = 1-2 and 100-1000 nM). This binding mimics ''specific'' ligand/receptor binding in that it is inhibited by 10 microM unlabeled 5-HT. This inhibition is not competitive, but rather is due to the prevention of (/sup 3/H)-5-HT breakdown by excess unlabeled 5-HT. Unlike genuine ligand/receptor binding, the binding of (/sup 3/H)-5-HT breakdown products is essentially irreversible and does not display a tissue distribution consistent with binding to authentic 5-HT receptors. (/sup 3/H)-5-HT decomposition can be eliminated by the inclusion of 0.05 to 5 mM ascorbic acid. At these concentrations ascorbic acid is not deleterious to reversible (/sup 3/H)-5-HT binding. When (/sup 3/H) 5-HT exposure to air occurs in the presence of brain membranes, the apparent antioxidant activity of brain membranes themselves affords protection against (/sup 3/H)-5-HT degradation equal to ascorbic acid. This protection is effective below final (/sup 3/H)-5-HT concentrations of 10 nM. Above 10 nM (/sup 3/H)-5-HT, addition of ascorbic acid or other antioxidants is necessary to avoid the occurrence of additional low affinity (apparent Kd = 15-2000 nM) binding sites that are specific but nonetheless irreversible. When care is taken to limit (/sup 3/H)-5-HT oxidation, the only reversible and saturable specific binding sites observed are of the 5-HT1 high affinity (Kd = 1-2 nM) type. Radioligand oxidation artifacts may be involved in previous reports of low affinity (Kd = 15-250 nM) (/sup 3/H)-5-HT binding sites in brain membrane preparations.

  18. Regional distribution of rat brain alpha/sub 1/-adrenergic receptors: correlation between (/sup 125/I)-heat membrane binding and in vitro autoradiography

    SciTech Connect

    Jones, L.S.; Miller, G.; Gauger, L.L.; Davis, J.N.

    1985-01-07

    (/sup 125/I)-HEAT has proven useful for in vitro autoradiography as a specific alpha/sub 1/-adrenergic radioligand. We compared the binding of (/sup 125/I)-HEAT to membranes from ten brain regions with the densitometric readings of these regions in autoradiographs. There was an excellent correlation between receptor numbers from membrane binding and relative optical densities from the autoradiography. The affinity of HEAT for binding to membranes from various regions was similar. The results of this direct comparison are further evidence that HEAT binds to alpha/sub 1/-adrenergic receptors in lightly fixed tissue sections. A further interesting observation is that in regions with a heterogeneous distribution of binding sites, membrane binding may not reflect the presence of a dense local population of receptors. 19 references, 3 figures, 1 table.

  19. F-actin binds to the cytoplasmic surface of ponticulin, a 17-kD integral glycoprotein from Dictyostelium discoideum plasma membranes

    PubMed Central

    1987-01-01

    F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gp17) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gp17 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N- ethylmaleimide. gp17 may be responsible for much of the actin-binding activity of plasma membranes since monovalent antibody fragments (Fab) directed primarily against gp17 inhibit actin-membrane binding by 96% in sedimentation assays. In contrast, Fab directed against cell surface determinants inhibit binding by only 0-10%. The actin-binding site of gp17 appears to be located on the cytoplasmic surface of the membrane since Fab against this protein continue to inhibit 96% of actin- membrane binding even after extensive adsorption against cell surfaces. gp17 is abundant in the plasma membrane, constituting 0.4-1.0% of the total membrane protein. A transmembrane orientation of gp17 is suggested since, in addition to the cytoplasmic localization of the actin-binding site, extracellular determinants of gp17 are identified. gp17 is surface-labeled by sulfo-N-hydroxy-succinimido-biotin, a reagent that cannot penetrate the cell membrane. Also, gp17 is glycosylated since it is specifically bound by the lectin, concanavalin A. We propose that gp17 is a major actin-binding protein that is important for connecting the plasma membrane to the underlying microfilament network. Therefore, we have named this protein "ponticulin" from the Latin word, ponticulus, which means small bridge. PMID:3312238

  20. Interaction of the two components of leukocidin from Staphylococcus aureus with human polymorphonuclear leukocyte membranes: sequential binding and subsequent activation.

    PubMed Central

    Colin, D A; Mazurier, I; Sire, S; Finck-Barbançon, V

    1994-01-01

    The sequential interaction between the two components S and F of leukocidin from Staphylococcus aureus and the membrane of human polymorphonuclear neutrophils has been investigated in the presence of 1 mM Ca2+. With 125I-labeled components, it has been shown that binding of the F component occurred only after binding of the S component. The kinetic constants of binding of both components were not statistically different (Kd, approximately 5 nM; Bm, approximately 35,000 molecules per cell), and both Hill coefficients were 1. The application of increasing concentrations of leukocidin provoked a dose-dependent secretion of the granule content, as determined by hexosaminidase and lysozyme activity measurements. Furthermore, the separate perfusion of S and F components on human polymorphonuclear neutrophils deposited on a filter induced secretion of the granules content only when the perfusion of the S component preceded that of the F component. We conclude, therefore, that (i) S-component binding is a prerequisite for F-component binding and for subsequent activation of polymorphonuclear neutrophils and (ii) there is a specific binding site for the S component in the plasma membrane. PMID:8039887

  1. Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom

    SciTech Connect

    Lambeau, G.; Barhanin, J.; Schweitz, H.; Qar, J.; Lazdunski, M. )

    1989-07-05

    Four new monochain phospholipases were purified from the Oxyuranus scutellatus (taipan) venom. Three of them were highly toxic when injected into mice brain. One of these neurotoxic phospholipases, OS2, was iodinated and used in binding experiments to demonstrate the presence of two families of specific binding sites in rat brain synaptic membranes. The affinities were exceptionally high, Kd1 = 1.5 +/- 0.5 pM and Kd2 = 45 +/- 10 pM, and the maximal binding capacities were Bmax 1 = 1 +/- 0.4 and Bmax 2 = 3 +/- 0.5 pmol/mg of protein. Both binding sites were sensitive to proteolysis and demonstrated to be located on proteins of Mr 85,000-88,000 and 36,000-51,000 by cross-linking and photoaffinity labeling techniques. The binding of {sup 125}I-OS2 to synaptic membranes was dependent on Ca2+ ions and enhanced by Zn2+ ions which inhibit phospholipase activity. Competition experiments have shown that, except for beta-bungarotoxin, a number of known toxic snake or bee phospholipases have very high affinities for the newly identified binding sites. A good correlation (r = 0.80) was observed between toxicity and affinity but not between phospholipase activity and affinity.

  2. Uncommon structural motifs dominate the antigen binding site in human autoantibodies reactive with basement membrane collagen.

    PubMed

    Foster, Mary H; Buckley, Elizabeth S; Chen, Benny J; Hwang, Kwan-Ki; Clark, Amy G

    2016-08-01

    Autoantibodies mediate organ destruction in multiple autoimmune diseases, yet their origins in patients remain poorly understood. To probe the genetic origins and structure of disease-associated autoantibodies, we engrafted immunodeficient mice with human CD34+ hematopoietic stem cells and immunized with the non-collagenous-1 (NC1) domain of the alpha3 chain of type IV collagen. This antigen is expressed in lungs and kidneys and is targeted by autoantibodies in anti-glomerular basement membrane (GBM) nephritis and Goodpasture syndrome (GPS), prototypic human organ-specific autoimmune diseases. Using Epstein Barr virus transformation and cell fusion, six human anti-alpha3(IV)NC1 collagen monoclonal autoantibodies (mAb) were recovered, including subsets reactive with human kidney and with epitopes recognized by patients' IgG. Sequence analysis reveals a long to exceptionally long heavy chain complementarity determining region3 (HCDR3), the major site of antigen binding, in all six mAb. Mean HCDR3 length is 25.5 amino acids (range 20-36), generated from inherently long DH and JH genes and extended regions of non-templated N-nucleotides. Long HCDR3 are suited to forming noncontiguous antigen contacts and to binding recessed, immunologically silent epitopes hidden from conventional antibodies, as seen with self-antigen crossreactive broadly neutralizing anti-HIV Ig (bnAb). The anti-alpha3(IV)NC1 collagen mAb also show preferential use of unmutated variable region genes that are enriched among human chronic lymphocytic leukemia antibodies that share features with natural polyreactive Ig. Our findings suggest unexpected relationships between pathogenic anti-collagen Ig, bnAb, and autoreactive Ig associated with malignancy, all of which arise from B cells expressing unconventional structural elements that may require transient escape from tolerance for successful expansion. PMID:27450516

  3. Bacterial binding protein-dependent permeases: characterization of distinctive signatures for functionally related integral cytoplasmic membrane proteins.

    PubMed

    Saurin, W; Köster, W; Dassa, E

    1994-06-01

    Bacterial binding protein-dependent transport systems belong to the superfamily of ABC transporters, which is widely distributed among living organisms. Their hydrophobic membrane proteins are the least characterized components. The primary structures of 61 integral membrane proteins from 35 uptake systems were compared in order to characterize a short conserved hydrophilic segment, with a consensus EAA---G---------I-LP, located approximately 100 residues from the C-terminus. Secondary structure predictions indicated that this conserved region might be formed by two amphipathic alpha-helices connected by a loop containing the invariant G residue. We classified the conserved motifs and found that membrane proteins from systems transporting structurally related substrates specifically display a greater number of identical residues in the conserved region. We determined a consensus for each class of membrane protein and showed that these can be considered as signatures. PMID:7934906

  4. Mechanisms of recognition and binding of α-TTP to the plasma membrane by multi-scale molecular dynamics simulations

    PubMed Central

    Lamprakis, Christos; Stocker, Achim; Cascella, Michele

    2015-01-01

    We used multiple sets of simulations both at the atomistic and coarse-grained level of resolution to investigate interaction and binding of α-tochoperol transfer protein (α-TTP) to phosphatidylinositol phosphate lipids (PIPs). Our calculations indicate that enrichment of membranes with such lipids facilitate membrane anchoring. Atomistic models suggest that PIP can be incorporated into the binding cavity of α-TTP and therefore confirm that such protein can work as lipid exchanger between the endosome and the plasma membrane. Comparison of the atomistic models of the α-TTP-PIPs complex with membrane-bound α-TTP revealed different roles for the various basic residues composing the basic patch that is key for the protein/ligand interaction. Such residues are of critical importance as several point mutations at their position lead to severe forms of ataxia with vitamin E deficiency (AVED) phenotypes. Specifically, R221 is main residue responsible for the stabilization of the complex. R68 and R192 exchange strong interactions in the protein or in the membrane complex only, suggesting that the two residues alternate contact formation, thus facilitating lipid flipping from the membrane into the protein cavity during the lipid exchange process. Finally, R59 shows weaker interactions with PIPs anyway with a clear preference for specific phosphorylation positions, hinting a role in early membrane selectivity for the protein. Altogether, our simulations reveal significant aspects at the atomistic scale of interactions of α-TTP with the plasma membrane and with PIP, providing clarifications on the mechanism of intracellular vitamin E trafficking and helping establishing the role of key residue for the functionality of α-TTP. PMID:26191529

  5. Heterodimeric Capping Protein from Arabidopsis Is a Membrane-Associated, Actin-Binding Protein1[W][OPEN

    PubMed Central

    Jimenez-Lopez, Jose C.; Wang, Xia; Kotchoni, Simeon O.; Huang, Shanjin; Szymanski, Daniel B.; Staiger, Christopher J.

    2014-01-01

    The actin cytoskeleton is a major regulator of cell morphogenesis and responses to biotic and abiotic stimuli. The organization and activities of the cytoskeleton are choreographed by hundreds of accessory proteins. Many actin-binding proteins are thought to be stimulus-response regulators that bind to signaling phospholipids and change their activity upon lipid binding. Whether these proteins associate with and/or are regulated by signaling lipids in plant cells remains poorly understood. Heterodimeric capping protein (CP) is a conserved and ubiquitous regulator of actin dynamics. It binds to the barbed end of filaments with high affinity and modulates filament assembly and disassembly reactions in vitro. Direct interaction of CP with phospholipids, including phosphatidic acid, results in uncapping of filament ends in vitro. Live-cell imaging and reverse-genetic analyses of cp mutants in Arabidopsis (Arabidopsis thaliana) recently provided compelling support for a model in which CP activity is negatively regulated by phosphatidic acid in vivo. Here, we used complementary biochemical, subcellular fractionation, and immunofluorescence microscopy approaches to elucidate CP-membrane association. We found that CP is moderately abundant in Arabidopsis tissues and present in a microsomal membrane fraction. Sucrose density gradient separation and immunoblotting with known compartment markers were used to demonstrate that CP is enriched on membrane-bound organelles such as the endoplasmic reticulum and Golgi. This association could facilitate cross talk between the actin cytoskeleton and a wide spectrum of essential cellular functions such as organelle motility and signal transduction. PMID:25201878

  6. Identification of concanavalin A receptors and galactose-binding proteins in purified plasma membranes of Dictyostelium discoideum.

    PubMed

    West, C M; McMahon, D

    1977-07-01

    Two techniques have been modified to provide simple means for the identification of molecules which bind concanavalin A (Con A). Crossed immunoelectrophoresis was altered by replacing antibody with Con A, and receptors were identified by the precipitin arcs which they produced. Con A, tagged with fluorescein isothiocyanate, was also diffused into prefixed sodium dodecyl sulfate (SDS)-polyacrylamide gels, and additional receptors identified by fluorescence. More than 35 molecules in the plasma membranes of the cellular slime mold Dictyostelium discoideum which bind Con A were identified with these techniques. At least 12 of these diminish and 12 increase in importance as receptors during differentiation of the cells from the vegetative to the preculmination stage of development. In the course of these experiments, it was possible to confirm the presence of the galactose-binding protein discoidin, in the plasma membrane, by electrophoresing membrane proteins into an agarose gel. This lectin regains its sugar-binding activity after denaturation and electrophoresis in SDS. PMID:559679

  7. Hypoxia in the androgen-dependent Shionogi model for prostate cancer at three stages.

    PubMed

    Skov, Kirsten; Adomat, Hans; Bowden, Mary; Dragowska, Wieslawa; Gleave, Martin; Koch, Cameron J; Woo, Janet; Yapp, Donald T T

    2004-11-01

    The objective of this study was to investigate a possible relationship between androgen status and hypoxia in the Shionogi murine prostate tumor model, which is widely used to study the effects of androgen withdrawal on hormone resistance and radiation response. Binding of the nitroimidazole hypoxia marker EF5 was assessed using the Cy3-tagged monoclonal antibody ELK3-51. Three hours after injection of EF5 (30 mg/kg), tumors from the following three stages were excised: androgen-dependent, regressed tumors 7 days after castration, and androgen-independent. Half of each tumor was disaggregated for analysis by flow cytometry and the remainder was flash frozen. Statistically significant differences (P < 0.01) were found between androgen-dependent, regressed and androgen-dependent tumors: approximately 30, approximately 2 and approximately 50% hypoxic cells, respectively. Frozen sections from androgen-dependent tumors exhibited highly variable EF5 binding; regressed tumors showed very little or no binding; each section from androgen-dependent tumors showed high levels and uniformly distributed binding of EF5. There was no correlation between the degree of hypoxia and tumor weight (P > 0.1). The results from this preliminary study indicate that hypoxia may play an important role with respect to the timing of irradiation in prostate cancer treatments and possibly may be a useful prognostic tool. In addition, hypoxia may also be relevant to progression in this disease after androgen ablation. PMID:15624309

  8. The Lipopeptide Antibiotic Paenibacterin Binds to the Bacterial Outer Membrane and Exerts Bactericidal Activity through Cytoplasmic Membrane Damage

    PubMed Central

    Huang, En

    2014-01-01

    Paenibacterin is a broad-spectrum lipopeptide antimicrobial agent produced by Paenibacillus thiaminolyticus OSY-SE. The compound consists of a cyclic 13-residue peptide and an N-terminal C15 fatty acyl chain. The mechanism of action of paenibacterin against Escherichia coli and Staphylococcus aureus was investigated in this study. The cationic lipopeptide paenibacterin showed a strong affinity for the negatively charged lipopolysaccharides (LPS) from the outer membrane of Gram-negative bacteria. Addition of LPS (100 μg/ml) completely eliminated the antimicrobial activity of paenibacterin against E. coli. The electrostatic interaction between paenibacterin and LPS may have displaced the divalent cations on the LPS network and thus facilitated the uptake of antibiotic into Gram-negative cells. Paenibacterin also damaged the bacterial cytoplasmic membrane, as evidenced by the depolarization of membrane potential and leakage of intracellular potassium ions from cells of E. coli and S. aureus. Therefore, the bactericidal activity of paenibacterin is attributed to disruption of the outer membrane of Gram-negative bacteria and damage of the cytoplasmic membrane of both Gram-negative and Gram-positive bacteria. Despite the evidence of membrane damage, this study does not rule out additional bactericidal mechanisms potentially exerted by paenibacterin. PMID:24561581

  9. Myristoylation of an inhibitory GTP-binding protein. alpha. subunit is essential for its membrane attachment

    SciTech Connect

    Jones, T.L.Z.; Simonds, W.F.; Merendino, J.J. Jr.; Brann, M.R.; Spiegel, A.M. )

    1990-01-01

    The authors transfected COS cells with cDNAs for the {alpha} subunits of stimulatory and inhibitory GTP-binding proteins, {alpha}{sub s} and {alpha}{sub i1}, respectively, and immunoprecipitated the metabolically labeled products with specific peptide antibodies. Cells were separated into particulate and soluble fractions before immunoprecipitation; ({sup 35}S)methionine-labeled {alpha}{sub s} and {alpha}{sub i} were both found primarily in the particulate fraction. ({sup 3}H)Myristate was incorporated into endogenous and transfected {alpha}{sub i} but could not be detected in {alpha}{sub s} even when it was overexpressed. They converted the second residue, glycine, of {alpha}{sub i1} into alanine by site-directed mutagenesis. Upon transfection of the mutant {alpha}{sub i1} into COS cells, the ({sup 35}S)methionine-labeled product was localized primarily to the soluble fraction, and, also unlike normal {alpha}{sub i1}, the mutant failed to incorporate ({sup 3}H)myristate. The unmyristoylated mutant {alpha}{sub i1} could still interact with the {beta}-{gamma} complex, since purified {beta}{gamma} subunits promoted pertussis toxin-catalyzed ADP-ribosylation of both the normal and mutant {alpha}{sub i1} subunits. These results indicate that myristoylation is critical for membrane attachment of {alpha}{sub i} but not {alpha}{sub s} subunits.

  10. Eisosomes Are Dynamic Plasma Membrane Domains Showing Pil1-Lsp1 Heteroligomer Binding Equilibrium

    PubMed Central

    Olivera-Couto, Agustina; Salzman, Valentina; Mailhos, Milagros; Digman, Michelle A.; Gratton, Enrico; Aguilar, Pablo S.

    2015-01-01

    Eisosomes are plasma membrane domains concentrating lipids, transporters, and signaling molecules. In the budding yeast Saccharomyces cerevisiae, these domains are structured by scaffolds composed mainly by two cytoplasmic proteins Pil1 and Lsp1. Eisosomes are immobile domains, have relatively uniform size, and encompass thousands of units of the core proteins Pil1 and Lsp1. In this work we used fluorescence fluctuation analytical methods to determine the dynamics of eisosome core proteins at different subcellular locations. Using a combination of scanning techniques with autocorrelation analysis, we show that Pil1 and Lsp1 cytoplasmic pools freely diffuse whereas an eisosome-associated fraction of these proteins exhibits slow dynamics that fit with a binding-unbinding equilibrium. Number and brightness analysis shows that the eisosome-associated fraction is oligomeric, while cytoplasmic pools have lower aggregation states. Fluorescence lifetime imaging results indicate that Pil1 and Lsp1 directly interact in the cytoplasm and within the eisosomes. These results support a model where Pil1-Lsp1 heterodimers are the minimal eisosomes building blocks. Moreover, individual-eisosome fluorescence fluctuation analysis shows that eisosomes in the same cell are not equal domains: while roughly half of them are mostly static, the other half is actively exchanging core protein subunits. PMID:25863055

  11. Lipid-Linked Oligosaccharides in Membranes Sample Conformations That Facilitate Binding to Oligosaccharyltransferase

    PubMed Central

    Kern, Nathan R.; Lee, Hui Sun; Wu, Emilia L.; Park, Soohyung; Vanommeslaeghe, Kenno; MacKerell, Alexander D.; Klauda, Jeffery B.; Jo, Sunhwan; Im, Wonpil

    2014-01-01

    Lipid-linked oligosaccharides (LLOs) are the substrates of oligosaccharyltransferase (OST), the enzyme that catalyzes the en bloc transfer of the oligosaccharide onto the acceptor asparagine of nascent proteins during the process of N-glycosylation. To explore LLOs’ preferred location, orientation, structure, and dynamics in membrane bilayers of three different lipid types (dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, and dioleoylphosphatidylcholine), we have modeled and simulated both eukaryotic (Glc3-Man9-GlcNAc2-PP-Dolichol) and bacterial (Glc1-GalNAc5-Bac1-PP-Undecaprenol) LLOs, which are composed of an isoprenoid moiety and an oligosaccharide, linked by pyrophosphate. The simulations show no strong impact of different bilayer hydrophobic thicknesses on the overall orientation, structure, and dynamics of the isoprenoid moiety and the oligosaccharide. The pyrophosphate group stays in the bilayer head group region. The isoprenoid moiety shows high flexibility inside the bilayer hydrophobic core, suggesting its potential role as a tentacle to search for OST. The oligosaccharide conformation and dynamics are similar to those in solution, but there are preferred interactions between the oligosaccharide and the bilayer interface, which leads to LLO sugar orientations parallel to the bilayer surface. Molecular docking of the bacterial LLO to a bacterial OST suggests that such orientations can enhance binding of LLOs to OST. PMID:25418169

  12. Cleavage of Nidogen-1 by Cathepsin S Impairs Its Binding to Basement Membrane Partners

    PubMed Central

    Sage, Juliette; Leblanc-Noblesse, Emmanuelle; Nizard, Carine; Sasaki, Takako; Schnebert, Sylvianne; Perrier, Eric; Kurfurst, Robin; Brömme, Dieter; Lalmanach, Gilles; Lecaille, Fabien

    2012-01-01

    Cathepsin S (catS), which is expressed in normal human keratinocytes and localized close to the dermal-epidermal junction (DEJ) degrades some of major basement membrane (BM) constituents. Among them, catS readily hydrolyzed in a time and dose dependent manner human nidogen-1 (nid-1) and nidogen-2, which are key proteins in the BM structure. CatS preferentially cleaved nid-1 at both acid and neutral pH. Hydrolysis of nid-1 was hampered in murine ctss−/− spleen lysates pretreated with inhibitors of other classes of proteases. Nid-1 was cleaved within its G2 and G3 globular domains that are both involved in interactions with other BM components. Binding assays with soluble and immobilized ligands indicated that catS altered the formation of complexes between nid-1 and other BM components. Assuming that the cleavage of nid-1 impairs its ability to crosslink with BM partners and perturbs the viscoelastic properties of BM matrix, these data indicate that catS may participate in BM proteolysis, in addition to already identified proteases. PMID:22952693

  13. Electrostatically induced recruitment of membrane peptides into clusters requires ligand binding at both interfaces.

    PubMed

    Antonenko, Yuri N; Horner, Andreas; Pohl, Peter

    2012-01-01

    Protein recruitment to specific membrane locations may be governed or facilitated by electrostatic attraction, which originates from a multivalent ligand. Here we explored the energetics of a model system in which this simple electrostatic recruitment mechanism failed. That is, basic poly-L-lysine binding to one leaflet of a planar lipid bilayer did not recruit the triply-charged peptide (O-Pyromellitylgramicidin). Clustering was only observed in cases where PLL was bound to both channel ends. Clustering was indicated (i) by the decreased diffusional PLL mobility D(PLL) and (ii) by an increased lifetime τ(PLL) of the clustered channels. In contrast, if PLL was bound to only one leaflet, neither D(PLL) nor τ(P) changed. Simple calculations suggest that electrostatic repulsion of the unbound ends prevented neighboring OPg dimers from approaching each other. We believe that a similar mechanism may also operate in cell signaling and that it may e.g. contribute to the controversial results obtained for the ligand driven dimerization of G protein-coupled receptors. PMID:23285199

  14. Electrostatically Induced Recruitment of Membrane Peptides into Clusters Requires Ligand Binding at Both Interfaces

    PubMed Central

    Pohl, Peter

    2012-01-01

    Protein recruitment to specific membrane locations may be governed or facilitated by electrostatic attraction, which originates from a multivalent ligand. Here we explored the energetics of a model system in which this simple electrostatic recruitment mechanism failed. That is, basic poly-L-lysine binding to one leaflet of a planar lipid bilayer did not recruit the triply-charged peptide (O-Pyromellitylgramicidin). Clustering was only observed in cases where PLL was bound to both channel ends. Clustering was indicated (i) by the decreased diffusional PLL mobility DPLL and (ii) by an increased lifetime τPLL of the clustered channels. In contrast, if PLL was bound to only one leaflet, neither DPLL nor τP changed. Simple calculations suggest that electrostatic repulsion of the unbound ends prevented neighboring OPg dimers from approaching each other. We believe that a similar mechanism may also operate in cell signaling and that it may e.g. contribute to the controversial results obtained for the ligand driven dimerization of G protein-coupled receptors. PMID:23285199

  15. Eisosomes are dynamic plasma membrane domains showing pil1-lsp1 heteroligomer binding equilibrium.

    PubMed

    Olivera-Couto, Agustina; Salzman, Valentina; Mailhos, Milagros; Digman, Michelle A; Gratton, Enrico; Aguilar, Pablo S

    2015-04-01

    Eisosomes are plasma membrane domains concentrating lipids, transporters, and signaling molecules. In the budding yeast Saccharomyces cerevisiae, these domains are structured by scaffolds composed mainly by two cytoplasmic proteins Pil1 and Lsp1. Eisosomes are immobile domains, have relatively uniform size, and encompass thousands of units of the core proteins Pil1 and Lsp1. In this work we used fluorescence fluctuation analytical methods to determine the dynamics of eisosome core proteins at different subcellular locations. Using a combination of scanning techniques with autocorrelation analysis, we show that Pil1 and Lsp1 cytoplasmic pools freely diffuse whereas an eisosome-associated fraction of these proteins exhibits slow dynamics that fit with a binding-unbinding equilibrium. Number and brightness analysis shows that the eisosome-associated fraction is oligomeric, while cytoplasmic pools have lower aggregation states. Fluorescence lifetime imaging results indicate that Pil1 and Lsp1 directly interact in the cytoplasm and within the eisosomes. These results support a model where Pil1-Lsp1 heterodimers are the minimal eisosomes building blocks. Moreover, individual-eisosome fluorescence fluctuation analysis shows that eisosomes in the same cell are not equal domains: while roughly half of them are mostly static, the other half is actively exchanging core protein subunits. PMID:25863055

  16. Binding Analyses of Bacillus thuringiensis Cry δ-Endotoxins Using Brush Border Membrane Vesicles of Ostrinia nubilalis

    PubMed Central

    Hua, Gang; Masson, Luke; Jurat-Fuentes, Juan Luis; Schwab, George; Adang, Michael J.

    2001-01-01

    Transgenic corn expressing the Bacillus thuringiensis Cry1Ab gene is highly insecticidal to Ostrinia nubilalis (European corn borer) larvae. We ascertained whether Cry1F, Cry9C, or Cry9E recognizes the Cry1Ab binding site on the O. nubilalis brush border by three approaches. An optical biosensor technology based on surface plasmon resonance measured binding of brush border membrane vesicles (BBMV) injected over a surface of immobilized Cry toxin. Preincubation with Cry1Ab reduced BBMV binding to immobilized Cry1Ab, whereas preincubation with Cry1F, Cry9C, or Cry9E did not inhibit BBMV binding. BBMV binding to a Cry1F-coated surface was reduced when vesicles were preincubated in Cry1F or Cry1Ab but not Cry9C or Cry9E. A radioligand approach measured 125I-Cry1Ab toxin binding to BBMV in the presence of homologous (Cry1Ab) and heterologous (Cry1Ac, Cry1F, Cry9C, or Cry9E) toxins. Unlabeled Cry1Ac effectively competed for 125I-Cry1Ab binding in a manner comparable to Cry1Ab itself. Unlabeled Cry9C and Cry9E toxins did not inhibit 125I-Cry1Ab binding to BBMV. Cry1F inhibited 125I-Cry1Ab binding at concentrations greater than 500 nM. Cry1F had low-level affinity for the Cry1Ab binding site. Ligand blot analysis identified Cry1Ab, Cry1Ac, and Cry1F binding proteins in BBMV. The major Cry1Ab signals on ligand blots were at 145 kDa and 154 kDa, but a strong signal was present at 220 kDa and a weak signal was present at 167 kDa. Cry1Ac and Cry1F binding proteins were detected at 220 and 154 kDa. Anti-Manduca sexta aminopeptidase serum recognized proteins of 145, 154, and 167 kDa, and anti-cadherin serum recognized the 220 kDa protein. We speculate that isoforms of aminopeptidase and cadherin in the brush border membrane serve as Cry1Ab, Cry1Ac, and Cry1F binding proteins. PMID:11157257

  17. ATP-binding cassette-like transporters are involved in the transport of lignin precursors across plasma and vacuolar membranes

    SciTech Connect

    Miao, Y.C.; Liu, C.

    2010-12-28

    Lignin is a complex biopolymer derived primarily from the condensation of three monomeric precursors, the monolignols. The synthesis of monolignols occurs in the cytoplasm. To reach the cell wall where they are oxidized and polymerized, they must be transported across the cell membrane. However, the molecular mechanisms underlying the transport process are unclear. There are conflicting views about whether the transport of these precursors occurs by passive diffusion or is an energized active process; further, we know little about what chemical forms are required. Using isolated plasma and vacuolar membrane vesicles prepared from Arabidopsis, together with applying different transporter inhibitors in the assays, we examined the uptake of monolignols and their derivatives by these native membrane vesicles. We demonstrate that the transport of lignin precursors across plasmalemma and their sequestration into vacuoles are ATP-dependent primary-transport processes, involving ATP-binding cassette-like transporters. Moreover, we show that both plasma and vacuolar membrane vesicles selectively transport different forms of lignin precursors. In the presence of ATP, the inverted plasma membrane vesicles preferentially take up monolignol aglycones, whereas the vacuolar vesicles are more specific for glucoconjugates, suggesting that the different ATP-binding cassette-like transporters recognize different chemical forms in conveying them to distinct sites, and that glucosylation of monolignols is necessary for their vacuolar storage but not required for direct transport into the cell wall in Arabidopsis.

  18. Molecular insight into the differential anti-androgenic activity of resveratrol and its natural analogs: in silico approach to understand biological actions.

    PubMed

    Chakraborty, Sandipan; Kumar, Avinash; Butt, Nasir A; Zhang, Liangfen; Williams, Raquema; Rimando, Agnes M; Biswas, Pradip K; Levenson, Anait S

    2016-04-26

    The androgen receptor (AR) is a therapeutic target for the treatment of prostate cancer. Androgen receptor reactivation during the androgen-independent stage of prostate cancer is mediated by numerous mechanisms including expression of AR mutants and splice variants that become non-responsive to conventional anti-androgenic agents. Resveratrol and its natural analogs exhibit varying degrees of anti-androgenic effects on tumor growth suppression in prostate cancer. However, the structural basis for the observed differential activity remains unknown. Here, anti-androgenic activities of resveratrol and its natural analogs, namely, pterostilbene, piceatannol and trimethoxy-resveratrol were studied in LNCaP cells expressing T877A mutant AR and atomistic simulations were employed to establish the structure activity relationship. Interestingly, essential hydrogen bonding contacts and the binding energies of resveratrol analogs with AR ligand binding domain (LBD), emerge as key differentiating factors for varying anti-androgenic action. Among all the analogs, pterostilbene exhibited strongest anti-androgenic activity and its binding energy and hydrogen bonding interactions pattern closely resembled pure anti-androgen, flutamide. Principal component analysis of our simulation studies revealed that androgenic compounds bind more strongly to AR LBD compared to anti-androgenic compounds and provide conformational stabilization of the receptor in essential subspace. The present study provides critical insight into the structure-activity relationship of the anti-androgenic action of resveratrol analogs, which can be translated further to design novel highly potent anti-androgenic stilbenes. PMID:27063447

  19. Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains[S

    PubMed Central

    Bhat, Hema Balakrishna; Kishimoto, Takuma; Abe, Mitsuhiro; Makino, Asami; Inaba, Takehiko; Murate, Motohide; Dohmae, Naoshi; Kurahashi, Atsushi; Nishibori, Kozo; Fujimori, Fumihiro; Greimel, Peter; Ishitsuka, Reiko; Kobayashi, Toshihide

    2013-01-01

    A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus, pleurotolysin (Ply)A, from the extract of edible mushroom Pleurotus eryngii, named PlyA2. Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-β-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding. Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes. PMID:23918047

  20. (/sup 3/H)Ethylketocyclazocine binding to mouse brain membranes: evidence for a kappa opioid receptor type

    SciTech Connect

    Garzon, J.; Sanchez-Blazquez, P.; Lee, N.M.

    1984-10-01

    The binding of the putative kappa agonist ethylketocyclazocine (EKC) to synaptosomal membranes of mouse brain was studied. This benzomorphan was able to bind to different opioid receptors. A portion of this binding was not inhibited by the agonist naloxone, even at high concentrations (10 microM). This population of receptors, to which opioate alkaloids and opiod peptides display very low affinity, is probably the sigma receptor. Another class of binding sites was identified by the simultaneous addition of the selective agonists Sandoz FK-33824 and D-Ala2-D-Leu5-enkephalin, which blocked the access of EKC to mu and delta opioid receptors, respectively, leaving a portion of naloxone-displaceable benzomorphan binding still detectable. Analysis of this remaining binding revealed a small population of receptors of high affinity, the kappa receptor. Therefore, EKC binds to the mu, delta, kappa and sigma receptors in the mouse brain, with similar affinities for the mu and kappa (0.22 and 0.15 nM). These results confirm the existence of a kappa opioid receptor type in the mouse brain.

  1. The liver X receptor agonist T0901317 acts as androgen receptor antagonist in human prostate cancer cells

    SciTech Connect

    Chuu, Chih-pin; Chen, Rou-Yu; Hiipakka, Richard A.; Kokontis, John M.; Warner, Karen V.; Xiang, Jialing; Liao, Shutsung . E-mail: sliao@uchicago.edu

    2007-06-01

    T0901317 is a potent non-steroidal synthetic liver X receptor (LXR) agonist. T0901317 blocked androgenic stimulation of the proliferation of androgen-dependent LNCaP 104-S cells and androgenic suppression of the proliferation of androgen-independent LNCaP 104-R2 cells, inhibited the transcriptional activation of an androgen-dependent reporter gene by androgen, and suppressed gene and protein expression of prostate specific antigen (PSA), a target gene of androgen receptor (AR) without affecting gene and protein expression of AR. T0901317 also inhibited binding of a radiolabeled androgen to AR, but inhibition was much weaker compared to the effect of the antiandrogens, bicalutamide and hydroxyflutamide. The LXR agonist T0901317, therefore, acts as an antiandrogen in human prostate cancer cells.

  2. Correlation between catecholamine secretion from bovine isolated chromaffin cells and [3H]-ouabain binding to plasma membranes

    PubMed Central

    Aunis, Dominique; García, Antonio G.

    1981-01-01

    1 Secretion of catecholamines (CA) evoked by ouabain, chlormadinone acetate (CMA), phenoxybenzamine (Pbz) and vanadate, four agents known to inhibit Na+, K+-dependent Mg2+-activated adenosine triphosphatase (ATPase) activity has been studied in suspensions of bovine isolated adrenal medullary cells. 2 Acetylcholine (ACh) evoked a 5 fold increase of the basal CA secretion from isolated cells suspended in oxygenated Krebs-bicarbonate solution kept at 27°C. Secretion was antagonized by Ca2+-deprivation or hexamethonium, indicating good functional viability of the cells. 3 Ouabain (10-7 to 10-4 M) evoked a progressive, dose-dependent release of CA from cell suspensions. Study of the time course of the secretory response for 2 h allowed the separation of two components in the secretory response at all doses studied: a slow initial component (0.011 pg/min CA) and a second faster component (0.032 pg/min CA). 4 CMA evoked a clear-cut CA secretory response. The ED50 for CMA was 10-4 M, as compared to 3 × 10-6 M for ouabain. Pbz and vanadate did not induce CA release. 5 [3H]-ouabain was taken up and bound to intact isolated cells by a non-saturable binding process. However, in semi-purified plasma membranes from bovine adrenal medulla a saturable specific [3H]-ouabain binding process was observed with a KD of 8.1 nM. Binding to the membranes was ATP-dependent and antagonized by K+. 6 [3H]-ouabain specific binding to membranes was antagonized by ouabain and CMA, but not by Pbz or vanadate; the ID50 for ouabain and CMA were 10-6 and 10-5 M respectively. 7 Ouabain partially inhibited, in a dose-dependent manner, Na+, K+-Mg2+ ATPase activity of the semi-purified plasma membranes. 8 The results demonstrate a good correlation between the ability of different drugs, known to inhibit ATPase activity, to displace [3H]-ouabain binding to adreno—medullary plasma membranes and their capacity to evoke a CA secretory response from isolated chromaffin cells. The data also suggest that

  3. Membrane Morphology Is Actively Transformed by Covalent Binding of the Protein Atg8 to PE-Lipids

    PubMed Central

    Knorr, Roland L.; Nakatogawa, Hitoshi; Ohsumi, Yoshinori; Lipowsky, Reinhard; Baumgart, Tobias; Dimova, Rumiana

    2014-01-01

    Autophagy is a cellular degradation pathway involving the shape transformation of lipid bilayers. During the onset of autophagy, the water-soluble protein Atg8 binds covalently to phosphatdylethanolamines (PEs) in the membrane in an ubiquitin-like reaction coupled to ATP hydrolysis. We reconstituted the Atg8 conjugation system in giant and nm-sized vesicles with a minimal set of enzymes and observed that formation of Atg8-PE on giant vesicles can cause substantial tubulation of membranes even in the absence of Atg12-Atg5-Atg16. Our findings show that ubiquitin-like processes can actively change properties of lipid membranes and that membrane crowding by proteins can be dynamically regulated in cells. Furthermore we provide evidence for curvature sorting of Atg8-PE. Curvature generation and sorting are directly linked to organelle shapes and, thus, to biological function. Our results suggest that a positive feedback exists between the ubiquitin-like reaction and the membrane curvature, which is important for dynamic shape changes of cell membranes, such as those involved in the formation of autophagosomes. PMID:25522362

  4. Intracellular kinetics of the androgen receptor shown by multimodal Image Correlation Spectroscopy (mICS)

    PubMed Central

    Chiu, Chi-Li; Patsch, Katherin; Cutrale, Francesco; Soundararajan, Anjana; Agus, David B.; Fraser, Scott E.; Ruderman, Daniel

    2016-01-01

    The androgen receptor (AR) pathway plays a central role in prostate cancer (PCa) growth and progression and is a validated therapeutic target. In response to ligand binding AR translocates to the nucleus, though the molecular mechanism is not well understood. We therefore developed multimodal Image Correlation Spectroscopy (mICS) to measure anisotropic molecular motion across a live cell. We applied mICS to AR translocation dynamics to reveal its multimodal motion. By integrating fluorescence imaging methods we observed evidence for diffusion, confined movement, and binding of AR within both the cytoplasm and nucleus of PCa cells. Our findings suggest that in presence of cytoplasmic diffusion, the probability of AR crossing the nuclear membrane is an important factor in determining the AR distribution between cytoplasm and the nucleus, independent of functional microtubule transport. These findings may have implications for the future design of novel therapeutics targeting the AR pathway in PCa. PMID:26936218

  5. Characterization of specific binding sites for (/sup 3/H)(d)-N-allylnormetazocine in rat brain membranes

    SciTech Connect

    Itzhak, Y.; Hiller, J.M.; Simon, E.J.

    1985-01-01

    Binding of (/sup 3/H)(d)-N-allylnormetazocine ((/sup 3/H)(d)-NANM) to rat brain membranes is stereospecific, reversible, and saturable (Bmax . 260 fmol/mg of protein) and manifests moderately high affinity (Kd . 20 nM). The rank order of potency among opioidbenzomorphans and phencyclidine (PCP) analogs for competition for (/sup 3/H)(d)-NANM-binding sites is as follows: (d)-NANM . PCP-3-OH greater than (d)-cyclazocine greater than N-ethylphenylcyclohexylamine greater than PCP greater than (l)-cyclazocine . dextrorphan greater than (d/l)-ethylketocyclazocine greater than (d/l)-bremazocine greater than (1)-NANM greater than 1-phenylcyclohexylamine greater than levorphanol. Other opioid ligands, relatively selective for each of the types of opioid binding sites other than sigma, such as morphine (mu), H-Tyr-D-Ala(Me)Phe-NH-CH2-OH (mu), D-Ala2-D-Leu5-enkephalin (delta), tifluadom (kappa), and U 50488 (kappa) as well as etorphine and naloxone were all unable to compete with (/sup 3/H)(d)-NANM for specific binding even at a concentration of 1 microM. Regional distribution studies of (/sup 3/H)(d)-NANM-binding sites show high density in the hippocampus, thalamus, hypothalamus, and amygdala and low density in cerebellum and nonfrontal neocortex membranes of the rat brain. These binding sites are very sensitive to protein-modifying enzymes and reagents such as trypsin and N-ethylmaleimide and to heat denaturation. These results provide direct biochemical evidence for the existence of distinct (d)-NANM-binding sites in rat brain.

  6. Mutational analysis of the androgen receptor gene in two Chinese families with complete androgen insensitivity syndrome

    PubMed Central

    WANG, SONG; XU, HAIKUN; AN, WEI; ZHU, DECHUN; LI, DEJUN

    2016-01-01

    Androgens are essential for normal male sex differentiation and are responsible for the normal development of male secondary sexual characteristics at puberty. The physiological effects of androgens are mediated by the androgen receptor (AR). Mutations in the AR gene are the most common cause of androgen insensitivity syndrome. The present study undertook a genetic analysis of the AR gene in two unrelated families affected by complete androgen insensitivity syndrome (CAIS) in China. In family 1, a previously reported nonsense mutation (G-to-A; p.W751X) was identified in exon 5 of the AR gene. In addition, a novel missense mutation was detected in exon 6 of the AR gene from family 2; this mutation resulted in a predicted amino acid change from phenylalanine to serine at codon 804 (T-to-C; p.F804S) in the ligand-binding domain (LBD) of AR. Computer simulation of the structural changes generated by the p.F804S substitution revealed marked conformational alterations in the hydrophobic core responsible for the stability and function of the AR-LBD. In conclusion, the present study identified two mutations from two unrelated Chinese families affected by CAIS. The novel mutation (p.F804S) may provide insights into the molecular mechanism underlying CAIS. Furthermore, it expands on the number of mutational hot spots in the international AR mutation database, which may be useful in the future for prenatal diagnosis and genetic counseling. PMID:27284311

  7. Characterization of a multiple endogenously expressed Adenosine triphosphate-Binding Cassette transporters using nuclear and cellular membrane affinity chromatography columns

    PubMed Central

    Khadeer, M.A.; Shimmo, R.; Wainer, I.W.; Moaddel, R.

    2014-01-01

    Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN229)) and (CMAC(LN229)), respectively. Pgp, MRP1and BCRP transporters co-immobilized on both columns was characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs 3.7μM), verapamil (0.6 vs 0.7μM) and prazosin (0.099 vs 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of 8 compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN229) column and decreased it (−5%) on the NMAC(LN229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences. PMID:24642394

  8. Binding of the cytoplasmic domain of CD28 to the plasma membrane inhibits Lck recruitment and signaling.

    PubMed

    Dobbins, Jessica; Gagnon, Etienne; Godec, Jernej; Pyrdol, Jason; Vignali, Dario A A; Sharpe, Arlene H; Wucherpfennig, Kai W

    2016-01-01

    The T cell costimulatory receptor CD28 is required for the full activation of naïve T cells and for the development and maintenance of Foxp3(+) regulatory T (Treg) cells. We showed that the cytoplasmic domain of CD28 was bound to the plasma membrane in resting cells and that ligand binding to CD28 resulted in its release. Membrane binding by the CD28 cytoplasmic domain required two clusters of basic amino acid residues, which interacted with the negatively charged inner leaflet of the plasma membrane. These same clusters of basic residues also served as interaction sites for Lck, a Src family kinase critical for CD28 function. This signaling complex was further stabilized by the Lck-mediated phosphorylation of CD28 Tyr(207) and the subsequent binding of the Src homology 2 (SH2) domain of Lck to this phosphorylated tyrosine. Mutation of the basic clusters in the CD28 cytoplasmic domain reduced the recruitment to the CD28-Lck complex of protein kinase Cθ (PKCθ), which serves as a key effector kinase in the CD28 signaling pathway. Consequently, mutation of either a basic cluster or Tyr(207) impaired CD28 function in mice as shown by the reduced thymic differentiation of FoxP3(+) Treg cells. On the basis of these results, we propose a previously undescribed model for the initiation of CD28 signaling. PMID:27460989

  9. Membrane binding of peptide models for early stages of amyloid formation: Lipid packing counts more than charge.

    PubMed

    Hoernke, Maria; Tassler, Stephanie; Koksch, Beate; Brezesinski, Gerald

    2016-06-01

    Amyloid formation is related to neurodegenerative diseases like Alzheimer's disease or Parkinson's disease. In the molecular onset of the diseases, soluble peptides adopt conformations that are rich in β-sheet and ultimately form aggregates. How this process is triggered or influenced by membrane binding, or how the membrane integrity is disturbed by the peptide binding and conformational transition is still under debate. In the present study, we systematically examine the effects of β-sheet prone model peptides on zwitterionic and negatively charged lipids in both mono- and bilayers and in various lipid phase states by infrared reflection absorption spectroscopy, grazing incidence X-ray diffraction, and small and wide angle X-ray scattering. No difference in the interaction of the peptides with zwitterionic or negatively charged lipids was observed. Furthermore, the interaction of β-sheet prone model peptides leaves the lipid structure largely unaffected. However, the lipid phase state decides upon the mode of interaction. Peptides insert into liquid-expanded layers and interact only with the head groups of liquid-condensed lipid layers. Using a zoo of complementary techniques and critically examining preparation procedures we are able to obtain an unambiguous picture of peptide binding to membranes. PMID:27134131

  10. High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall.

    PubMed

    Canut, H; Carrasco, A; Galaud, J P; Cassan, C; Bouyssou, H; Vita, N; Ferrara, P; Pont-Lezica, R

    1998-10-01

    The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD--the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (Kd1 approximately 1 nM, and Kd2 approximately 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments. PMID:9807828

  11. IL-6 and IL-8 enhance factor H binding to the cell membranes

    PubMed Central

    POPEK, SYLWIA; KAPKA-SKRZYPCZAK, LUCYNA; SAWICKI, KRZYSZTOF; WOLIŃSKA, EWA; SKRZYPCZAK, MACIEJ; CZAJKA, MAGDALENA

    2016-01-01

    The aim of the present study was to assess the role of interleukin (IL)-6 and IL-8 on the expression of fluid-phase complement inhibitor, factor H (FH), and FH-like protein 1 (FHL-1), in the A2780 ovarian carcinoma cell line. This cell line does not normally produce IL-6, however, is IL-6 responsive due to the presence of receptor for IL-6. The presence of FH and FHL-1 in the cell lysates was confirmed by western blotting. The levels of FH and FHL-1 in the medium were determined by enzyme-linked immunosorbent assay. To evaluate gene expression, reverse transcription-quantitative polymerase chain reaction was performed. The cellular localization of FH and FHL-1 in ovarian cancer cells was assessed by immunofluorescence. The present study revealed that FH, contrary to FHL-1, was secreted by ovarian cancer cells, however, this process was independent of IL stimulation. No significant differences were observed in the concentration of FH in the control cells, when compared with the samples treated with IL-6/IL-8. The results of western blotting revealed that the protein expression levels of FH and FHL-1 were not regulated by IL-6 and IL-8 in a dose-dependent manner. Immunofluorescence analysis confirmed that the A2780 ovarian cancer cell line expressed both membrane bound and intracellular forms of FH and FHL-1. The present data revealed that the A2780 cells expressed and secreted FH protein and are also able to bind FH and FHL-1. This may influence the efficiency of complement mediated immunotherapy. PMID:27035765

  12. A Major Fraction of Glycosphingolipids in Model and Cellular Cholesterol-containing Membranes Is Undetectable by Their Binding Proteins*

    PubMed Central

    Mahfoud, Radhia; Manis, Adam; Binnington, Beth; Ackerley, Cameron; Lingwood, Clifford A.

    2010-01-01

    Glycosphingolipids (GSLs) accumulate in cholesterol-enriched cell membrane domains and provide receptors for protein ligands. Lipid-based “aglycone” interactions can influence GSL carbohydrate epitope presentation. To evaluate this relationship, Verotoxin binding its receptor GSL, globotriaosyl ceramide (Gb3), was analyzed in simple GSL/cholesterol, detergent-resistant membrane vesicles by equilibrium density gradient centrifugation. Vesicles separated into two Gb3/cholesterol-containing populations. The lighter, minor fraction (<5% total GSL), bound VT1, VT2, IgG/IgM mAb anti-Gb3, HIVgp120 or Bandeiraea simplicifolia lectin. Only IgM anti-Gb3, more tolerant of carbohydrate modification, bound both vesicle fractions. Post-embedding cryo-immuno-EM confirmed these results. This appears to be a general GSL-cholesterol property, because similar receptor-inactive vesicles were separated for other GSL-protein ligand systems; cholera toxin (CTx)-GM1, HIVgp120-galactosyl ceramide/sulfatide. Inclusion of galactosyl or glucosyl ceramide (GalCer and GlcCer) rendered VT1-unreactive Gb3/cholesterol vesicles, VT1-reactive. We found GalCer and GlcCer bind Gb3, suggesting GSL-GSL interaction can counter cholesterol masking of Gb3. The similar separation of Vero cell membrane-derived vesicles into minor “binding,” and major “non-binding” fractions when probed with VT1, CTx, or anti-SSEA4 (a human GSL stem cell marker), demonstrates potential physiological relevance. Cell membrane GSL masking was cholesterol- and actin-dependent. Cholesterol depletion of Vero and HeLa cells enabled differential VT1B subunit labeling of “available” and “cholesterol-masked” plasma membrane Gb3 pools by fluorescence microscopy. Thus, the model GSL/cholesterol vesicle studies predicted two distinct membrane GSL formats, which were demonstrated within the plasma membrane of cultured cells. Cholesterol masking of most cell membrane GSLs may impinge many GSL receptor functions. PMID

  13. Binding kinetics of Clostridium difficile toxins A and B to intestinal brush border membranes from infant and adult hamsters

    SciTech Connect

    Rolfe, R.D. )

    1991-04-01

    This study was undertaken to determine if the relative resistance of neonates and infants to Clostridium difficile-associated intestinal disease can be related to age-dependent differences in intestinal receptors for C. difficile toxins A and B. Brush border membranes (BBMs) from the small intestines of adult and infant hamsters were examined for their ability to bind radiolabeled toxins A and B. (125I)toxin A bound to both infant and adult hamster BBMs at physiological temperature, whereas (125I)toxin B did not bind to the BBMs under any of the conditions examined. The number of (125I)toxin A molecules bound at saturation was approximately 4 x 10(10) per micrograms of membrane protein for adult BBMs and 1 x 10(11) per micrograms of membrane protein for infant BBMs. Scatchard plot analysis suggested the presence of a single class of toxin A binding sites on both infant and adult hamster BBMs. Maximal binding capacity and Kd values were 0.63 pmol/mg of protein and 66.7 nM, respectively, for the infant BBMs, and 0.24 pmol/mg of protein and 27 nM, respectively, for the adult BBMs. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses of extracted BBM proteins revealed differences in the proteins of infant and adult BBMs. However, there were not any detectable differences in the protein bands which bound (125I)toxin A between infant and adult hamsters. The results from these investigations indicate that differences in the binding kinetics of toxins A and/or B to infant and adult hamster BBMs do not account for the observed differences in their susceptibility to C. difficile-associated intestinal disease.

  14. Carbohydrate-Derived Amphiphilic Macromolecules: A Biophysical Structural Characterization and Analysis of Binding Behaviors to Model Membranes

    PubMed Central

    Martin, Adriana A. T.; Tomasini, Michael; Kholodovych, Vladyslav; Gu, Li; Sommerfeld, Sven Daniel; Uhrich, Kathryn E.; Murthy, N. Sanjeeva; Welsh, William J.; Moghe, Prabhas V.

    2015-01-01

    The design and synthesis of enhanced membrane-intercalating biomaterials for drug delivery or vascular membrane targeting is currently challenged by the lack of screening and prediction tools. The present work demonstrates the generation of a Quantitative Structural Activity Relationship model (QSAR) to make a priori predictions. Amphiphilic macromolecules (AMs) “stealth lipids” built on aldaric and uronic acids frameworks attached to poly(ethylene glycol) (PEG) polymer tails were developed to form self-assembling micelles. In the present study, a defined set of novel AM structures were investigated in terms of their binding to lipid membrane bilayers using Quartz Crystal Microbalance with Dissipation (QCM-D) experiments coupled with computational coarse-grained molecular dynamics (CG MD) and all-atom MD (AA MD) simulations. The CG MD simulations capture the insertion dynamics of the AM lipophilic backbones into the lipid bilayer with the PEGylated tail directed into bulk water. QCM-D measurements with Voigt viscoelastic model analysis enabled the quantitation of the mass gain and rate of interaction between the AM and the lipid bilayer surface. Thus, this study yielded insights about variations in the functional activity of AM materials with minute compositional or stereochemical differences based on membrane binding, which has translational potential for transplanting these materials in vivo. More broadly, it demonstrates an integrated computational-experimental approach, which can offer a promising strategy for the in silico design and screening of therapeutic candidate materials. PMID:25855953

  15. Discriminating Lipid- from Protein-Calcium Binding To Understand the Interaction between Recoverin and Phosphatidylglycerol Model Membranes.

    PubMed

    Potvin-Fournier, Kim; Lefèvre, Thierry; Picard-Lafond, Audrey; Marcotte, Catherine; Dufresne, Caroline; Cantin, Line; Salesse, Christian; Auger, Michèle

    2016-06-21

    Recoverin is a protein involved in the phototransduction cascade by regulating the activity of rhodopsin kinase through a calcium-dependent binding process at the surface of rod outer segment disk membranes. Understanding how calcium modulates these interactions and how it interacts with anionic lipid membranes is necessary to gain insights into the function of recoverin. In this work, infrared spectroscopy allowed us to show that the availability of calcium to recoverin is modulated by the presence of complexes involving phosphatidylglycerol (PG), which in turn regulates its interactions with this negatively charged lipid. Calcium can indeed be sequestered into strongly bound complexes with PG and is thus sparingly available to recoverin. The thermal stability of recoverin then decreases, which results in weakened interactions with PG. By contrast, when calcium is fully available to recoverin, the protein is thermally stable, indicating that it binds two calcium ions, which results in favorable interactions with negatively charged lipids. Consequently, the protein induces an increase in the chain-melting phase transition temperature of PG, which is indicative of an enhanced lipid chain packing resulting from the peripheral location of the protein. The secondary structure of recoverin is not affected by its interactions with anionic membrane lipids. Similar results have been obtained with saturated and unsaturated anionic lipids. This work shows that the recruitment of recoverin at the surface of anionic lipid membranes is dependent on the availability of calcium. PMID:27240971

  16. Membrane Binding of MinE Allows for a Comprehensive Description of Min-Protein Pattern Formation

    PubMed Central

    Bonny, Mike; Fischer-Friedrich, Elisabeth; Loose, Martin; Schwille, Petra; Kruse, Karsten

    2013-01-01

    The rod-shaped bacterium Escherichia coli selects the cell center as site of division with the help of the proteins MinC, MinD, and MinE. This protein system collectively oscillates between the two cell poles by alternately binding to the membrane in one of the two cell halves. This dynamic behavior, which emerges from the interaction of the ATPase MinD and its activator MinE on the cell membrane, has become a paradigm for protein self-organization. Recently, it has been found that not only the binding of MinD to the membrane, but also interactions of MinE with the membrane contribute to Min-protein self-organization. Here, we show that by accounting for this finding in a computational model, we can comprehensively describe all observed Min-protein patterns in vivo and in vitro. Furthermore, by varying the system's geometry, our computations predict patterns that have not yet been reported. We confirm these predictions experimentally. PMID:24339757

  17. Binding affinity of amyloid oligomers to cellular membranes is a generic indicator of cellular dysfunction in protein misfolding diseases.

    PubMed

    Evangelisti, Elisa; Cascella, Roberta; Becatti, Matteo; Marrazza, Giovanna; Dobson, Christopher M; Chiti, Fabrizio; Stefani, Massimo; Cecchi, Cristina

    2016-01-01

    The conversion of peptides or proteins from their soluble native states into intractable amyloid deposits is associated with a wide range of human disorders. Misfolded protein oligomers formed during the process of aggregation have been identified as the primary pathogenic agents in many such conditions. Here, we show the existence of a quantitative relationship between the degree of binding to neuronal cells of different types of oligomers formed from a model protein, HypF-N, and the GM1 content of the plasma membranes. In addition, remarkably similar behavior is observed for oligomers of the Aβ42 peptide associated with Alzheimer's disease. Further analysis has revealed the existence of a linear correlation between the level of the influx of Ca(2+) across neuronal membranes that triggers cellular damage, and the fraction of oligomeric species bound to the membrane. Our findings indicate that the susceptibility of neuronal cells to different types of misfolded oligomeric assemblies is directly related to the extent of binding of such oligomers to the cellular membrane. PMID:27619987

  18. Membrane Thinning Due to Antimicrobial Peptide Binding: An Atomic Force Microscopy Study of MSI-78 in Lipid Bilayers

    PubMed Central

    Mecke, Almut; Lee, Dong-Kuk; Ramamoorthy, Ayyalusamy; Orr, Bradford G.; Banaszak Holl, Mark M.

    2005-01-01

    The interaction of an antimicrobial peptide, MSI-78, with phospholipid bilayers has been investigated using atomic force microscopy, circular dichroism, and nuclear magnetic resonance (NMR). Binding of amphipathic peptide helices with their helical axis parallel to the membrane surface leads to membrane thinning. Atomic force microscopy of supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers in the presence of MSI-78 provides images of the membrane thinning process at a high spatial resolution. This data reveals that the membrane thickness is not reduced uniformly over the entire bilayer area. Instead, peptide binding leads to the formation of distinct domains where the bilayer thickness is reduced by 1.1 ± 0.2 nm. The data is interpreted using a previously published geometric model for the structure of the peptide-lipid domains. In this model, the peptides reside at the hydrophilic-hydrophobic boundary in the lipid headgroup region, which leads to an increased distance between lipid headgroups. This picture is consistent with concentration-dependent 31P and 2H NMR spectra of MSI-78 in mechanically aligned DMPC bilayers. Furthermore, 2H NMR experiments on DMPC-d54 multilamellar vesicles indicate that the acyl chains of DMPC are highly disordered in the presence of the peptide as is to be expected for the proposed structure of the peptide-lipid assembly. PMID:16183881

  19. Nanoscale Distribution of Sulfonic Acid Groups Determines Structure and Binding of Water in Nafion Membranes.

    PubMed

    Ling, Xiao; Bonn, Mischa; Parekh, Sapun H; Domke, Katrin F

    2016-03-14

    The connection between the nanoscale structure of two chemically equivalent, yet morphologically distinct Nafion fuel-cell membranes and their macroscopic chemical properties is demonstrated. Quantification of the chemical interactions between water and Nafion reveals that extruded membranes have smaller water channels with a reduced sulfonic acid head group density compared to dispersion-cast membranes. As a result, a disproportionally large amount of non-bulk water molecules exists in extruded membranes, which also exhibit larger proton conductivity and larger water mobility compared to cast membranes. The differences in the physicochemical properties of the membranes, that is, the chemical constitution of the water channels and the local water structure, and the accompanying differences in macroscopic water and proton transport suggest that the chemistry of nanoscale channels is an important, yet largely overlooked parameter that influences the functionality of fuel-cell membranes. PMID:26895211

  20. Clinical, cytogenetic and molecular analysis of androgen insensitivity syndromes from south Indian cohort and detection and in-silico characterization of androgen receptor gene mutations.

    PubMed

    V G, Abilash; S, Radha; K M, Marimuthu; K, Thangaraj; S, Arun; S, Nishu; A, Mohana Priya; J, Meena; D, Anuradha

    2016-01-30

    Rare cases of 9 complete androgen insensitivity syndromes, 9 cases of partial androgen insensitivity syndromes and equal number of male control samples were selected for this study. Few strong variations in clinical features were noticed; Giemsa banded metaphase revealed a 46,XY karyotype and the frequency of chromosome aberrations were significantly higher when compared with control samples. DNA sequence analysis of the androgen receptor gene of androgen insensitivity syndromes revealed three missense mutations - c.C1713>G resulting in the replacement of a highly conserved histidine residue with glutamine p.(His571Glu) in DNA-binding domain, c.A1715>G resulting in the replacement of a highly conserved tyrosine residue with cysteine p.(Tyr572Cys) in DNA-binding domain and c.G2599>A resulting in the replacement of a highly conserved valine residue with methionine p.(Val867Met) in ligand-binding domain of androgen receptor gene respectively. The heterozygous type of mutations c.C1713>G and c.G2599>A observed in mothers of the patients for familial cases concluding that the mutation was inherited from the mother. The novel mutation c.C1713>G is reported first time in androgen insensitivity syndrome. In-silico analysis of mutations observed in androgen receptor gene of androgen insensitivity syndrome predicted that the substitution at Y572C and V867M could probably disrupt the protein structure and function. PMID:26688387

  1. The Juxtamembrane Linker of Full-length Synaptotagmin 1 Controls Oligomerization and Calcium-dependent Membrane Binding*

    PubMed Central

    Lu, Bin; Kiessling, Volker; Tamm, Lukas K.; Cafiso, David S.

    2014-01-01

    Synaptotagmin 1 (Syt1) is the calcium sensor for synchronous neurotransmitter release. The two C2 domains of Syt1, which may mediate fusion by bridging the vesicle and plasma membranes, are connected to the vesicle membrane by a 60-residue linker. Here, we use site-directed spin labeling and a novel total internal reflection fluorescence vesicle binding assay to characterize the juxtamembrane linker and to test the ability of reconstituted full-length Syt1 to interact with opposing membrane surfaces. EPR spectroscopy demonstrates that the majority of the linker interacts with the membrane interface, thereby limiting the extension of the C2A and C2B domains into the cytoplasm. Pulse dipolar EPR spectroscopy provides evidence that purified full-length Syt1 is oligomerized in the membrane, and mutagenesis indicates that a glycine zipper/GXXXG motif within the linker helps mediate oligomerization. The total internal reflection fluorescence-based vesicle binding assay demonstrates that full-length Syt1 that is reconstituted into supported lipid bilayers will capture vesicles containing negatively charged lipid in a Ca2+-dependent manner. Moreover, the rate of vesicle capture increases with Syt1 density, and mutations in the GXXXG motif that inhibit oligomerization of Syt1 reduce the rate of vesicle capture. This work demonstrates that modifications within the 60-residue linker modulate both the oligomerization of Syt1 and its ability to interact with opposing bilayers. In addition to controlling its activity, the oligomerization of Syt1 may play a role in organizing proteins within the active zone of membrane fusion. PMID:24973220

  2. Large structure rearrangement of colicin ia channel domain after membrane binding from 2D 13C spin diffusion NMR.

    PubMed

    Luo, Wenbin; Yao, Xiaolan; Hong, Mei

    2005-05-01

    One of the main mechanisms of membrane protein folding is by spontaneous insertion into the lipid bilayer from the aqueous environment. The bacterial toxin, colicin Ia, is one such protein. To shed light on the conformational changes involved in this dramatic transfer from the polar to the hydrophobic milieu, we carried out 2D magic-angle spinning (13)C NMR experiments on the water-soluble and membrane-bound states of the channel-forming domain of colicin Ia. Proton-driven (13)C spin diffusion spectra of selectively (13)C-labeled protein show unequivocal attenuation of cross-peaks after membrane binding. This attenuation can be assigned to distance increases but not reduction of the diffusion coefficient. Analysis of the statistics of the interhelical and intrahelical (13)C-(13)C distances in the soluble protein structure indicates that the observed cross-peak reduction is well correlated with a high percentage of short interhelical contacts in the soluble protein. This suggests that colicin Ia channel domain becomes open and extended upon membrane binding, thus lengthening interhelical distances. In comparison, cross-peaks with similar intensities between the two states are dominated by intrahelical contacts in the soluble state. This suggests that the membrane-bound structure of colicin Ia channel domain may be described as a "molten globule", in which the helical secondary structure is retained while the tertiary structure is unfolded. This study demonstrates that (13)C spin diffusion NMR is a valuable tool for obtaining qualitative long-range distance constraints on membrane protein folding. PMID:15853348

  3. Androgen receptor: structure, role in prostate cancer and drug discovery

    PubMed Central

    Tan, MH Eileen; Li, Jun; Xu, H Eric; Melcher, Karsten; Yong, Eu-leong

    2015-01-01

    Androgens and androgen receptors (AR) play a pivotal role in expression of the male phenotype. Several diseases, such as androgen insensitivity syndrome (AIS) and prostate cancer, are associated with alterations in AR functions. Indeed, androgen blockade by drugs that prevent the production of androgens and/or block the action of the AR inhibits prostate cancer growth. However, resistance to these drugs often occurs after 2–3 years as the patients develop castration-resistant prostate cancer (CRPC). In CRPC, a functional AR remains a key regulator. Early studies focused on the functional domains of the AR and its crucial role in the pathology. The elucidation of the structures of the AR DNA binding domain (DBD) and ligand binding domain (LBD) provides a new framework for understanding the functions of this receptor and leads to the development of rational drug design for the treatment of prostate cancer. An overview of androgen receptor structure and activity, its actions in prostate cancer, and how structural information and high-throughput screening have been or can be used for drug discovery are provided herein. PMID:24909511

  4. Bee venom phospholipase A2 as a membrane-binding vector for cell surface display or internalization of soluble proteins.

    PubMed

    Babon, Aurélie; Wurceldorf, Thibault; Almunia, Christine; Pichard, Sylvain; Chenal, Alexandre; Buhot, Cécile; Beaumelle, Bruno; Gillet, Daniel

    2016-06-15

    We showed that bee venom phospholipase A2 can be used as a membrane-binding vector to anchor to the surface of cells a soluble protein fused to its C-terminus. ZZ, a two-domain derivative of staphylococcal protein A capable of binding constant regions of antibodies was fused to the C-terminus of the phospholipase or to a mutant devoid of enzymatic activity. The fusion proteins bound to the surface of cells and could themselves bind IgGs. Their fate depended on the cell type to which they bound. On the A431 carcinoma cell line the proteins remained exposed on the cell surface. In contrast, on human dendritic cells the proteins were internalized into early endosomes. PMID:26253725

  5. Properties of the deoxycholate-solubilized HeLa cell plasma membrane receptor for binding group B coxsackieviruses.

    PubMed Central

    Krah, D L; Crowell, R L

    1985-01-01

    Physical and chemical properties of deoxycholate-solubilized HeLa cell plasma membrane receptors for binding group B coxsackieviruses were determined. Receptors eluted from Sepharose 4B with an apparent molecular weight of 275,000 and sedimented with an S value of between 14.7 and 4.9 and a buoyant density of 1.06 to 1.10 g/cm3. Virus-binding activity was destroyed after treatment with proteases, glycosidases, and periodate but was unaffected by lipases or reducing or alkylating agents. Additionally, lectins, including concanavalin A, adsorbed receptors and inhibited virus attachment. The composite data suggested that glycoprotein is an integral part of the receptors for binding virus. PMID:2983096

  6. Characterization of the 3,3',5-triiodo-L-thyronine-binding site on plasma membranes from human placenta

    SciTech Connect

    Alderson, R.; Pastan, I.; Cheng, S.

    1985-06-01

    The binding of (/sup 125/I)T3 to sites on human placenta plasma membranes was characterized, and the binding site was solubilized after affinity labeling with N-bromoacetyl-(/sup 125/I)T3 (BrAc(/sup 125/I)T3). Two classes of T3-binding sites were detected. One class has a high affinity (K /sub d/ = 2.0nM) and a low capacity (approximately 320 fmol/mg protein); the other has a low affinity (K /sub k/ = 18.5 microM) and a high capacity (approximately 2.2 pmol/mg protein). The binding sites were found to be specific for T3 in that other thyroid hormone analogs (D-T3, rT3, D-T4, and L-T4) were less effective or ineffective in displacing the bound (/sup 125/I)T3. The affinity labeling ligand BrAc(/sup 125/I)T3 was found to specifically label a protein with an apparent mol wt of 65,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The BrAc(/sup 125/I)T3-labeled protein was solubilized with 2 mM 3-(( 3-cholamidopropyl)dimethylammonio)1-propane sulfonate. The apparent mol wt of the labeled protein was between 140,000 and 150,000 by Sephadex-G-200 gel filtration. These data demonstrate that a high affinity binding site specific for T3 is present on plasma membranes from human placenta and that the binding site is a protein, most likely a dimer, with a native mol wt between 140,000 and 150,000.

  7. Effect of surface and membrane potentials on IAA (indoleactic acid) uptake and binding by zucchini membrane vesicles

    SciTech Connect

    Clark, K.A.; Goldsmith, M.H.M.

    1986-08-01

    The polar transport of the endogenous hormone controlling extension growth of plant cells, indoleacetic acid (IAA), is thought to depend on transmembrane pH and electrical gradients resulting in part from the action of proton ATPases in the plasma membrane. Elements of this transport process are permeation of the membrane by the undissociated lipophilic indoleacetic acid (IAAH) from the acidic apoplast, followed by dissociation of the weak acid and accumulation of the IAA anion (IAA/sup -/) in the alkaline cytoplasm; a saturable symport of IAA/sup -/ with one or more protons; a carrier-mediated efflux of IAA/sup -/ down a considerable electrochemical gradient. The efflux is greater from the basal than the apical end of cells and is thought to be responsible for the overall polarity of the process. This step is also the site of action of napthylphthalamic acid (NPA) and herbicides that inhibit polar transport but stimulate net accumulation of auxin by tissues and cells. We are using membrane vesicles as a simplified system for studying the mechanisms involved in the transport and accumulation of auxin. In particular, we are interested in determining the involvement of the transmembrane pH (pH/sub o/ < pH/sub i/) and voltage gradients (K/sup +/ diffusion potential, (K/sup +/)/sub in/ > (K/sup +/)/sub out/) in IAA uptake. 19 refs., 6 figs.

  8. Structural Basis for Membrane Binding and Remodeling by the Exomer Secretory Vesicle Cargo Adaptor

    PubMed Central

    Paczkowski, Jon E.; Fromme, J. Christopher

    2014-01-01

    Summary Cargo adaptor subunits of vesicle coat protein complexes sort transmembrane proteins to distinct membrane compartments in eukaryotic cells. The exomer complex is the only cargo adaptor known to sort proteins at the trans-Golgi network into secretory vesicles. Exomer function is regulated by the Arf1 GTPase, a master regulator of trafficking at the Golgi. We report the structure of exomer bound to two copies of Arf1. Exomer interacts with each Arf1 molecule via two surfaces; one is a non-canonical interface that regulates GTP hydrolysis. The structure uncovers an unexpected membrane-proximal hydrophobic element that exomer uses in cooperation with Arf1 to remodel membranes. Given the constrained motion of the exomer hinge region, we envision that exomer dynamically positions multiple membrane insertion elements to drive membrane fission. In contrast to other known cargo adaptors, exomer therefore couples two functions, cargo sorting and membrane fission, into a single complex. PMID:25203211

  9. Intrinsically disordered linker and plasma membrane-binding motif sort Ist2 and Ssy1 to junctions.

    PubMed

    Kralt, Annemarie; Carretta, Marco; Mari, Muriel; Reggiori, Fulvio; Steen, Anton; Poolman, Bert; Veenhoff, Liesbeth M

    2015-02-01

    Membrane junctions or contact sites are close associations of lipid bilayers of heterologous organelles. Ist2 is an endoplasmic reticulum (ER)-resident transmembrane protein that mediates associations between the plasma membrane (PM) and the cortical ER (cER) in baker's yeast. We asked the question what structure in Ist2 bridges the up to 30 nm distance between the PM and the cER and we noted that the region spacing the transmembrane domain from the cortical sorting signal interacting with the PM is predicted to be intrinsically disordered (ID). In Ssy1, a protein that was not previously described to reside at membrane junctions, we recognized a domain organization similar to that in Ist2. We found that the localization of both Ist2 and Ssy1 at the cell periphery depends on the presence of a PM-binding domain, an ID linker region of sufficient length and a transmembrane domain that most probably resides in the ER. We show for the first time that an ID amino acid domain bridges adjacent heterologous membranes. The length and flexibility of ID domains make them uniquely eligible for spanning large distances, and we suggest that this domain structure occurs more frequently in proteins that mediate the formation of membrane contact sites. PMID:25409870

  10. ``Lock and key mechanism'' for ligand binding with adrenergic receptors and the arising mechanical effects on the cell membrane

    NASA Astrophysics Data System (ADS)

    Lunghi, Laura; Deseri, Luca

    2013-03-01

    Chemicals hitting the surface of cell aggregates are known to give arise to cyclic Adenosine Mono Phosphate (cAMP), a second messenger that transduces inside the cell the effects of species that cannot get through the cell membrane. Ligands bind to a specific receptor following the so called ``lock and key mechanism'' (beta)-adrenergic receptors are proteins embedded in the lipid bilayer characterized by seven transmembrane helices. Thinning and thickening in cell membranes may be initiated by conformational changes of some of three of the seven domains above. The cell response is linked to the coupling of chemical, conformational and mechanical effects. Part of the cAMP remains intracellular, whereas the remaining fractions migrates outside the cell due to membrane transporters. A new Helmholtz free energy, accounting for receptor and transporter densities, receptor conformation field and membrane elasticity is investigated. It is shown how the density of active receptors is directly related to the conformation field and it enters the resulting balance equation for the membrane stress. Balance laws for fluxes of transporters and receptors, coupled with the former because of the outgoing cAMP flux caused by the transporters, as well as for the diffusive powers must be supplied. The Center for Nonlinear Analysis through the NSF Grant No. DMS-0635983 is gratefully acknowledged.

  11. Steroid hormone modulation of 3H-prostaglanding E1 binding to bovine corpus leteum cell membranes.

    PubMed

    Rao, C V

    1975-04-01

    The specific binding of 3H-prostaglandin E1 (3H-PGE1) to bovine corpus luteum cell membranes was not affected by cholesterol or various progestins at concentrations of up to 9.0x10-minus-6M. At concentrations above 2.5 x 10-minus-6M; estrone, 17beta-estradiol (but not 17alpha-estradiol or 17beta-estradiol glucuronide), estroil, equilin, D-equilenin, 17-ethynyl estradiol, diethylstilbestrol, cortisol, corticosterone, deoxycorticosterone and aldosterone inhibited specific binding of 3H-PGE1. On the other hand, testosterone and dihydrotestosterone (DHT) (but not androstenedione) significantly enhanced 3H-PGE1 binding. These findings permitted the following correlations between steroid structure and modulation of 3H-PGE1 binding: steroids with a free phenolic ring and a 17beta-hydroxyl or 17-keto group or C-21 steroids with a C-20 ketone and a C-21 hydroxy group decrease, whereas C-19 steroids with a C-17 hydroxy group enhance specific binding of 3H-PGE1. PGE receptors are heterogeneous with respect to affinity for 3H-PGE1. The steroids that decreased 3H-PGE1 binding caused a lowering to a complete loss of low affinity PGE receptors. Steroids that increased 3H-PGE1 binding caused appearance of new low affinity PGE receptors. Association rate constants for 3H-PGE1 binding were decreased by 17beta-estradiol (61%) and increased by DHT (59%). PMID:168618

  12. Opioid binding to rat and guinea-pig neural membranes in the presence of physiological cations at 37 degrees C

    SciTech Connect

    Werling, L.L.; Zarr, G.D.; Brown, S.R.; Cox, B.M.

    1985-06-01

    The authors have identified mu, delta and kappa opioid binding sites in four types of neural membranes under conditions which include physiological concentrations of ions and an incubation temperature of 37 degrees C. They hypothesize that binding parameters determined under these conditions should be more directly comparable with physiological experiments than parameters obtained under conditions of low ionic concentration and at low temperature. By using either a radioligand which is selective for a single type of opioid binding site or a relatively nonselective radioligand in the presence of an unlabeled selective ligand, they have isolated binding to single populations of sites. Saturation and displacement data were analyzed with the aid of a computerized nonlinear curve fitting program. (/sup 3/H)Tyr-D-Ala-Gly-(Me)Phe-Gly-ol bound to a single population of sites with the characteristics of mu receptors, as determined by saturation and displacement analysis. Binding to the mu site represented 70% of the total specific opioid binding in rat brain, but only 20 to 30% in guinea-pig tissues. (/sup 3/H)(D-Ala2-D-Leu5)enkephalin bound almost equally well to mu and delta sites, but the delta site could be examined by the inclusion of unlabeled Tyr-D-Ala-Gly-(Me)Phe-Gly-ol in the incubations. (/sup 3/H)Ethylketocyclazocine bound mu and kappa sites, and Tyr-D-Ala-Gly-(Me)Phe-Gly-ol was also used to block the mu component in experiments in which they studied kappa binding. Binding to kappa sites represented 50 to 60% of the total in guinea-pig tissues, but less than 20% in rat brain.

  13. Selective membrane disruption by the cyclotide kalata B7: complex ions and essential functional groups in the phosphatidylethanolamine binding pocket.

    PubMed

    Strömstedt, Adam A; Kristiansen, Per Eugen; Gunasekera, Sunithi; Grob, Nathalie; Skjeldal, Lars; Göransson, Ulf

    2016-06-01

    The cyclic cystine knot plant peptides called cyclotides are active against a wide variety of organisms. This is primarily achieved through membrane binding and disruption, in part deriving from a high affinity for phosphatidylethanolamine (PE) lipids. Some cyclotides, such as kalata B7 (kB7), form complexes with divalent cations in a pocket associated with the tyrosine residue at position 15 (Tyr15). In the current work we explore the effect of cations on membrane leakage caused by cyclotides kB1, kB2 and kB7, and we identify a functional group that is essential for PE selectivity. The presence of PE-lipids in liposomes increased the membrane permeabilizing potency of the cyclotides, with the potency of kB7 increasing by as much as 740-fold. The divalent cations Mn(2+), Mg(2+) and Ca(2+) had no apparent effect on PE selectivity. However, amino acid substitutions in kB7 proved that Tyr15 is crucial for PE-selective membrane permeabilization on various liposome systems. Although the tertiary structure of kB7 was maintained, as reflected by the NMR solution structure, mutating Tyr into Ser at position 15 resulted in substantially reduced PE selectivity. Ala substitution at the same position produced a similar reduction in PE selectivity, while substitution with Phe maintained high selectivity. We conclude that the phenyl ring in Tyr15 is critical for the high PE selectivity of kB7. Our results suggest that PE-binding and divalent cation coordination occur in the same pocket without adverse effects of competitive binding for the phospholipid. PMID:26878982

  14. Identification of an additional class of C3-binding membrane proteins of human peripheral blood leukocytes and cell lines.

    PubMed Central

    Cole, J L; Housley, G A; Dykman, T R; MacDermott, R P; Atkinson, J P

    1985-01-01

    Proteins binding the third component of complement (C3) were isolated by affinity chromatography from surface-labeled solubilized membranes of human peripheral blood cells and cell lines. The isolated molecules were subjected to NaDodSO4/PAGE, and autoradiographs of these gels indicated that C3-binding proteins could be divided into three groups based on Mr: (i) gp200, an approximately 200,000 Mr molecule previously identified as the C3b/C4b receptor or CR1; (ii) gp140, an approximately 140,000 Mr molecule previously identified as the C3d receptor or CR2; and (iii) gp45-70, a heretofore unrecognized group of 45,000-70,000 Mr C3-binding molecules. The cell distribution, Mr, antigenic cross-reactivity, and specificity of gp45-70 were examined. Erythrocytes have no detectable gp45-70, but all leukocyte populations examined possess this group of molecules. On neutrophils and mononuclear phagocytes, CR1 is the predominant C3-binding glycoprotein, but gp45-70 is present on both cell populations and on macrophage and neutrophil cell lines. B plus null cells, chronic lymphocytic leukemia cells, and an Epstein-Barr virus-transformed B-cell line possess CR1, CR2, and gp45-70. On T cells and T-cell lines gp45-70 is the predominant or, in some cases, the only C3-binding protein isolated. gp45-70 is structurally characterized as a broad band or doublet with a mean Mr that is slightly different for each cell population. gp45-70 binds iC3, C3b, and C4b, but not C3d, indicating that the binding region is probably within the C3c portion of C3b. A polyclonal antibody to CR1 and monoclonal antibodies to CR1 and CR2 do not immunoprecipitate gp45-70. While gp45-70 has not been previously characterized on human cells, a C3b-binding glycoprotein of similar Mr is present on rabbit alveolar macrophages. We conclude that gp45-70 is an additional group of membrane proteins present on human leukocytes that possess ligand-binding activity for C3b. Images PMID:3871945

  15. Identification of an additional class of C3-binding membrane proteins of human peripheral blood leukocytes and cell lines.

    PubMed

    Cole, J L; Housley, G A; Dykman, T R; MacDermott, R P; Atkinson, J P

    1985-02-01

    Proteins binding the third component of complement (C3) were isolated by affinity chromatography from surface-labeled solubilized membranes of human peripheral blood cells and cell lines. The isolated molecules were subjected to NaDodSO4/PAGE, and autoradiographs of these gels indicated that C3-binding proteins could be divided into three groups based on Mr: (i) gp200, an approximately 200,000 Mr molecule previously identified as the C3b/C4b receptor or CR1; (ii) gp140, an approximately 140,000 Mr molecule previously identified as the C3d receptor or CR2; and (iii) gp45-70, a heretofore unrecognized group of 45,000-70,000 Mr C3-binding molecules. The cell distribution, Mr, antigenic cross-reactivity, and specificity of gp45-70 were examined. Erythrocytes have no detectable gp45-70, but all leukocyte populations examined possess this group of molecules. On neutrophils and mononuclear phagocytes, CR1 is the predominant C3-binding glycoprotein, but gp45-70 is present on both cell populations and on macrophage and neutrophil cell lines. B plus null cells, chronic lymphocytic leukemia cells, and an Epstein-Barr virus-transformed B-cell line possess CR1, CR2, and gp45-70. On T cells and T-cell lines gp45-70 is the predominant or, in some cases, the only C3-binding protein isolated. gp45-70 is structurally characterized as a broad band or doublet with a mean Mr that is slightly different for each cell population. gp45-70 binds iC3, C3b, and C4b, but not C3d, indicating that the binding region is probably within the C3c portion of C3b. A polyclonal antibody to CR1 and monoclonal antibodies to CR1 and CR2 do not immunoprecipitate gp45-70. While gp45-70 has not been previously characterized on human cells, a C3b-binding glycoprotein of similar Mr is present on rabbit alveolar macrophages. We conclude that gp45-70 is an additional group of membrane proteins present on human leukocytes that possess ligand-binding activity for C3b. PMID:3871945

  16. Trio engagement via plasma membrane phospholipids and the myristoyl moiety governs HIV-1 matrix binding to bilayers

    PubMed Central

    Vlach, Jiri; Saad, Jamil S.

    2013-01-01

    Localization of the HIV type-1 (HIV-1) Gag protein on the plasma membrane (PM) for virus assembly is mediated by specific interactions between the N-terminal myristoylated matrix (MA) domain and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. The PM bilayer is highly asymmetric, and this asymmetry is considered crucial in cell function. In a typical mammalian cell, the inner leaflet of the PM is enriched in phosphatidylserine (PS) and phosphatidylethanolamine (PE) and contains minor populations of phosphatidylcholine (PC) and PI(4,5)P2. There is strong evidence that efficient binding of HIV-1 Gag to membranes is sensitive not only to lipid composition and net negative charge, but also to the hydrophobic character of the acyl chains. Here, we show that PS, PE, and PC interact directly with MA via a region that is distinct from the PI(4,5)P2 binding site. Our NMR data also show that the myristoyl group is readily exposed when MA is bound to micelles or bicelles. Strikingly, our structural data reveal a unique binding mode by which the 2′-acyl chain of PS, PE, and PC lipids is buried in a hydrophobic pocket whereas the 1′-acyl chain is exposed. Sphingomyelin, a major lipid localized exclusively on the outer layer of the PM, does not bind to MA. Our findings led us to propose a trio engagement model by which HIV-1 Gag is anchored to the PM via the 1′-acyl chains of PI(4,5)P2 and PS/PE/PC and the myristoyl group, which collectively bracket a basic patch projecting toward the polar leaflet of the membrane. PMID:23401539

  17. The Borrelia afzelii outer membrane protein BAPKO_0422 binds human factor-H and is predicted to form a membrane-spanning β-barrel

    PubMed Central

    Dyer, Adam; Brown, Gemma; Stejskal, Lenka; Laity, Peter R.; Bingham, Richard J.

    2015-01-01

    The deep evolutionary history of the Spirochetes places their branch point early in the evolution of the diderms, before the divergence of the present day Proteobacteria. As a spirochete, the morphology of the Borrelia cell envelope shares characteristics of both Gram-positive and Gram-negative bacteria. A thin layer of peptidoglycan, tightly associated with the cytoplasmic membrane, is surrounded by a more labile outer membrane (OM). This OM is rich in lipoproteins but with few known integral membrane proteins. The outer membrane protein A (OmpA) domain is an eight-stranded membrane-spanning β-barrel, highly conserved among the Proteobacteria but so far unknown in the Spirochetes. In the present work, we describe the identification of four novel OmpA-like β-barrels from Borrelia afzelii, the most common cause of erythema migrans (EM) rash in Europe. Structural characterization of one these proteins (BAPKO_0422) by SAXS and CD indicate a compact globular structure rich in β-strand consistent with a monomeric β-barrel. Ab initio molecular envelopes calculated from the scattering profile are consistent with homology models and demonstrate that BAPKO_0422 adopts a peanut shape with dimensions 25×45 Å (1 Å=0.1 nm). Deviations from the standard C-terminal signature sequence are apparent; in particular the C-terminal phenylalanine residue commonly found in Proteobacterial OM proteins is replaced by isoleucine/leucine or asparagine. BAPKO_0422 is demonstrated to bind human factor H (fH) and therefore may contribute to immune evasion by inhibition of the complement response. Encoded by chromosomal genes, these proteins are highly conserved between Borrelia subspecies and may be of diagnostic or therapeutic value. PMID:26181365

  18. FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets

    PubMed Central

    Begonja, Antonija Jurak; Pluthero, Fred G.; Suphamungmee, Worawit; Giannini, Silvia; Christensen, Hilary; Leung, Richard; Lo, Richard W.; Nakamura, Fumihiko; Lehman, William; Plomann, Markus; Hoffmeister, Karin M.; Kahr, Walter H. A.; Hartwig, John H.

    2015-01-01

    Bin-Amphiphysin-Rvs (BAR) and Fes-CIP4 homology BAR (F-BAR) proteins generate tubular membrane invaginations reminiscent of the megakaryocyte (MK) demarcation membrane system (DMS), which provides membranes necessary for future platelets. The F-BAR protein PACSIN2 is one of the most abundant BAR/F-BAR proteins in platelets and the only one reported to interact with the cytoskeletal and scaffold protein filamin A (FlnA), an essential regulator of platelet formation and function. The FlnA-PACSIN2 interaction was therefore investigated in MKs and platelets. PACSIN2 associated with FlnA in human platelets. The interaction required FlnA immunoglobulin-like repeat 20 and the tip of PACSIN2 F-BAR domain and enhanced PACSIN2 F-BAR domain membrane tubulation in vitro. Most human and wild-type mouse platelets had 1 to 2 distinct PACSIN2 foci associated with cell membrane GPIbα, whereas Flna-null platelets had 0 to 4 or more foci. Endogenous PACSIN2 and transfected enhanced green fluorescent protein-PACSIN2 were concentrated in midstage wild-type mouse MKs in a well-defined invagination of the plasma membrane reminiscent of the initiating DMS and dispersed in the absence of FlnA binding. The DMS appeared less well defined, and platelet territories were not readily visualized in Flna-null MKs. We conclude that the FlnA-PACSIN2 interaction regulates membrane tubulation in MKs and platelets and likely contributes to DMS formation. PMID:25838348

  19. 68Ga-Labeled Anti-Prostate-Specific Membrane Antigen Peptide as Marker for Androgen Deprivation Therapy Response in Prostate Cancer.

    PubMed

    Schlenkhoff, Carl Diedrich; Gaertner, Florian; Essler, Markus; Hauser, Stefan; Ahmadzadehfar, Hojjat

    2016-05-01

    Prostate cancer was diagnosed in a 71-year-old man with an elevated prostate-specific antigen. The CT of the abdomen showed multiple para-aortal lymph nodes, and thus, a Ga anti-prostate-specific membrane antigen (PSMA-11) PET/CT was initiated, which showed, aside from the prostate cancer and multiple iliacal and para-aortal lymph node metastases, an increased tracer uptake in a lymph node left cervical. According to this advanced disease, a palliative therapy with GnRH agonist was initiated. A second PSMA-11 PET/CT was performed 4 months later, which showed a very good response; thus, additional radiation of the pelvis and the draining lymphatic system was performed. PMID:26859213

  20. INTERACTION OF INORGANIC MERCURY SALTS WITH MODEL AND RED CELL MEMBRANES: IMPORTANCE OF LIPID BINDING SALTS

    EPA Science Inventory

    The effect induced by two mercury salts, HgCl2 and Hg(NO3)2, on the thermotropic properties of PS model membranes (multilamellar vesicles) and rat red cell membranes was investigated employing 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence polarization. ercury(II) interacts wit...

  1. Differential binding of fibroblast growth factor-2 and -7 to basement membrane heparan sulfate: comparison of normal and abnormal human tissues.

    PubMed Central

    Friedl, A.; Chang, Z.; Tierney, A.; Rapraeger, A. C.

    1997-01-01

    Fibroblast growth factors (FGFs) play multiple roles during development and in adult tissues as paracrine regulators of growth and differentiation. FGFs signal through transmembrane receptor tyrosine kinases, but heparan sulfate is also required for signaling by members of the FGF family. In addition, heparan sulfate may be involved in determining tissue distribution of FGFs. Using biotinylated FGF-2 and FGF-7 (KGF) as probes, we have identified specific interactions between FGFs and heparan sulfates in human tissues. Both FGF species bind to tissue mast cells and to epithelial cell membranes. Binding to basement membrane heparan sulfate is tissue source dependent and specific. Although FGF-2 strongly binds to basement membrane heparan sulfate in skin and most other tissue sites examined, FGF-7 fails to bind to basement membrane heparan sulfate in most locations. However, in subendothelial matrix in blood vessels and in the basement membrane of a papillary renal cell carcinoma, strong FGF-7 binding is seen. In summary, distinct and specific affinities of heparan sulfates for different FGFs were identified that may affect growth factor activation and local distribution. Heparan sulfate may have a gatekeeper function to either restrict or permit diffusion of heparin-binding growth factors across the basement membrane. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 PMID:9094999

  2. Role of portal region lysine residues in electrostatic interactions between heart fatty acid binding protein and phospholipid membranes.

    PubMed

    Herr, F M; Aronson, J; Storch, J

    1996-01-30

    The structure of heart fatty acid binding protein (HFABP) is a flattened beta-barrel comprising 10 antiparallel beta-sheets capped by two alpha-helical segments. The helical cap region is hypothesized to behave as a portal "lid" for the entry and release of ligand from the binding pocket. The transfer of fatty acid from HFABP is thought to occur via effective collisional interactions with membranes, and these interactions are enhanced when transfer is to membranes of net negative charge, thus implying that specific basic residues on the surface of HFABP may govern the transfer process [Wootan, M. G., & Storch, J. (1994) J. Biol. Chem. 269, 10517-10523]. To directly examine the role of charged lysine residues on the HFABP surface in specific interactions with membranes, chemical modification and selective mutagenesis of HFABP were used. All surface lysine residues were neutralized by acetylation of recombinant HFABP with acetic anhydride. In addition, seven mutant HFABPs were generated that resulted in charge alterations in five distinct sites of HFABP. Modification of the protein did not significantly alter the structural or ligand binding properties of HFABP, as assessed by circular dichroism, fluorescence quantum yield, and ligand binding analyses. By using a resonance energy transfer assay, transfer of 2-(9-anthroyloxy)palmitate (2AP) from acetylated HFABP to membranes was significantly slower than transfer from native HFABP. In addition, in distinct contrast to transfer from native protein, the 2AP transfer rate from acetylated HFABP was not increased to acceptor membranes of increased negative charge. Transfer of 2AP from HFABP mutants involving K22, located on alpha-helix I (alpha-I) of the helical cap region, was 3-fold slower than transfer from wild-type protein, whereas rates from a mutant involving the K59 residue, located on the beta 2-turn of the barrel near the helical cap, were 2-fold faster than those of wild type. A double mutant involving K22 and K

  3. The physiological and pharmacological basis for the ergogenic effects of androgens in elite sports.

    PubMed

    Choong, Karen; Lakshman, Kishore M; Bhasin, Shalender

    2008-05-01

    Androgen doping in power sports is undeniably rampant worldwide. There is strong evidence that androgen administration in men increases skeletal muscle mass, maximal voluntary strength and muscle power. However, we do not have good experimental evidence to support the presumption that androgen administration improves physical function or athletic performance. Androgens do not increase specific force or whole body endurance measures. The anabolic effects of testosterone on the skeletal muscle are mediated through androgen receptor signaling. Testosterone promotes myogenic differentiation of multipotent mesenchymal stem cells and inhibits their differentiation into the adipogenic lineage. Testosterone binding to androgen receptor induces a conformational change in androgen receptor protein, causing it to associate with beta-catenin and TCF-4 and activate downstream Wnt target genes thus promoting myogenic differentiation. The adverse effects of androgens among athletes and recreational bodybuilders are under reported and include acne, deleterious changes in the cardiovascular risk factors, including a marked decrease in plasma high-density lipoproteins (HDL) cholesterol level, suppression of spermatogenesis resulting in infertility, increase in liver enzymes, hepatic neoplasms, mood and behavioral disturbances, and long term suppression of the endogenous hypothalamic-pituitary-gonadal axis. Androgens are often used in combination with other drugs which may have serious adverse events of their own. In spite of effective methods for detecting androgen doping, the policies for screening of athletes are highly variable in different countries and organizations and even existing policies are not uniformly enforced. PMID:18385897

  4. Androgens alter T-cell immunity by inhibiting T-helper 1 differentiation.

    PubMed

    Kissick, Haydn T; Sanda, Martin G; Dunn, Laura K; Pellegrini, Kathryn L; On, Seung T; Noel, Jonathan K; Arredouani, Mohamed S

    2014-07-01

    The hormonal milieu influences immune tolerance and the immune response against viruses and cancer, but the direct effect of androgens on cellular immunity remains largely uncharacterized. We therefore sought to evaluate the effect of androgens on murine and human T cells in vivo and in vitro. We found that murine androgen deprivation in vivo elicited RNA expression patterns conducive to IFN signaling and T-cell differentiation. Interrogation of mechanism showed that testosterone regulates T-helper 1 (Th1) differentiation by inhibiting IL-12-induced Stat4 phosphorylation: in murine models, we determined that androgen receptor binds a conserved region within the phosphatase, Ptpn1, and consequent up-regulation of Ptpn1 then inhibits IL-12 signaling in CD4 T cells. The clinical relevance of this mechanism, whereby the androgen milieu modulates CD4 T-cell differentiation, was ascertained as we found that androgen deprivation reduced expression of Ptpn1 in CD4 cells from patients undergoing androgen deprivation therapy for prostate cancer. Our findings, which demonstrate a clinically relevant mechanism by which androgens inhibit Th1 differentiation of CD4 T cells, provide rationale for targeting androgens to enhance CD4-mediated immune responses in cancer or, conversely, for modulating androgens to mitigate CD4 responses in disorders of autoimmunity. PMID:24958858

  5. Structural Basis of Pullulanase Membrane Binding and Secretion Revealed by X-Ray Crystallography, Molecular Dynamics and Biochemical Analysis.

    PubMed

    East, Alexandra; Mechaly, Ariel E; Huysmans, Gerard H M; Bernarde, Cédric; Tello-Manigne, Diana; Nadeau, Nathalie; Pugsley, Anthony P; Buschiazzo, Alejandro; Alzari, Pedro M; Bond, Peter J; Francetic, Olivera

    2016-01-01

    The Klebsiella lipoprotein pullulanase (PulA) is exported to the periplasm, triacylated, and anchored via lipids in the inner membrane (IM) prior to its transport to the bacterial surface through a type II secretion system (T2SS). X-Ray crystallography and atomistic molecular dynamics (MD) simulations of PulA in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) model membrane provided an unprecedented molecular view of an N-terminal unstructured tether and the IM lipoprotein retention signal, and revealed novel interactions with the IM via N-terminal immunoglobulin-like domains in PulA. An efficiently secreted nonacylated variant (PulANA) showed similar peripheral membrane association during MD simulations, consistent with the binding of purified PulANA to liposomes. Remarkably, combined X-ray, MD, and functional studies identified a novel subdomain, Ins, inserted in the α-amylase domain, which is required for PulA secretion. Available data support a model in which PulA binding to the IM promotes interactions with the T2SS, possibly via the Ins subdomain. PMID:26688215

  6. Macrophage Membrane Potential Changes Associated with γ 2b/γ 1 Fc Receptor-Ligand Binding

    NASA Astrophysics Data System (ADS)

    Young, John Ding-E; Unkeless, Jay C.; Kaback, H. Ronald; Cohn, Zanvil A.

    1983-03-01

    We have studied the effects of specific ligands of the receptor for the IgG Fc fragment (FcR) on the membrane potential (Δ Psi ) of the macrophage cell line J774 by the [3H]tetraphenylphosphonium ion equilibration technique. We observe a membrane depolarization with binding of FcR ligands that is dependent on the degree of receptor crosslinking. Binding of the FcR by monovalent ligands is not sufficient to induce a significant drop in Δ Psi , but a sustained depolarization lasting ≈ 20 min occurs with insoluble multivalent ligands. This FcR-mediated depolarization can be inhibited by substitution of Na+ from the cell incubation medium with monovalent choline cation, indicating that depolarization is due to Na+ influx into the cell. The extracellular Ca2+ does not play a significant role in membrane depolarization. The depolarization response is not triggered by monoclonal antibodies directed against three other major macrophage surface antigens. The cell depolarization mediated by FcR ligands is followed by a prolonged hyperpolarization that can be partially blocked by ouabain and quinine, indicating that the hyperpolarization response is a result of a combination of a Na+, K+-ATPase activity and a Ca2+-activated K+ conductance. These data support our hypothesis that the mouse macrophage IgG FcR is a ligand-dependent ion channel.

  7. Interaction between the inner nuclear membrane lamin B receptor and the heterochromatic methyl binding protein, MeCP2

    SciTech Connect

    Guarda, Alessia; Bolognese, Fabrizio; Bonapace, Ian Marc; Badaracco, Gianfranco

    2009-07-01

    The nuclear membrane has an important role for the dynamic regulation of the genome, besides the well-established cytoskeletal function. The nuclear lamina is emerging as an important player in the organization of the position and functional state of interphase chromosomes. Epigenetic modifications such as DNA methylation and histone modifications are required for genome reprogramming during development, tissue-specific gene expression and global gene silencing. The Methyl-CpG binding protein MeCP2 binds methyl-CpG dinucleotides in the mammalian genome and functions as a transcriptional repressor in vivo by interacting with Sin3A, thereby recruiting histone deacetylases (HDAC). MeCP2 also mediates the formation of higher-order chromatin structures contributing to determine the architectural organization of the nucleus. In this paper, we show that MeCP2 interacts in vitro and in vivo with the inner nuclear membrane protein LBR and that the unstructured aminoacidic sequence linking the MBD and TRD domains of MeCP2 is responsible for this association. The formation of an LBR-MeCP2 protein complex might help providing a molecular explanation to the distribution of part of the heterochromatin at the nuclear periphery linked to inner membrane.

  8. A membrane-bound form of glutamate dehydrogenase possesses an ATP-dependent high-affinity microtubule-binding activity.

    PubMed Central

    Rajas, F; Rousset, B

    1993-01-01

    We previously identified a 50 kDa membrane protein which bound to in vitro assembled microtubules [Mithieux and Rousset (1989) J. Biol. Chem. 264, 4664-4668]. This protein exhibited the expected properties for mediating the ATP-dependent association of vesicles with microtubules [Mithieux, Audebet and Rousset (1988) Biochim. Biophys. Acta 969, 121-130]. The 50 kDa membrane protein (MP50), initially extracted in very low amount from isolated pig thyroid lysosomes/endosomes, has now been purified from membrane preparations of crude vesicle fractions from pig liver and brain. MP50 was isolated from detergent-solubilized membrane protein by affinity chromatography on immobilized ATP; 3-5 mg of MP50 was obtained from 100 g of liver tissue. Phase partitioning in Triton X-114 indicated that MP50 is a peripheral membrane protein. Radioiodinated liver MP50 bound to microtubules assembled in vitro. The binding was inhibited by ATP (Ki = 0.76 mM) and displaced by unlabelled liver or brain MP50. Equilibrium binding studies yielded KD values of 1.8 x 10(-7) M. By N-terminal amino acid sequence analysis, MP50 was identified as glutamate dehydrogenase (GDH), by comparison of V8 protease peptide maps of MP50 with purified liver GDH. Liver MP50 exhibited a low GDH activity; 4-5 units/mg compared with 18 and 34 units/mg for purified bovine and rat liver GDH respectively. Bovine and rat liver GDH yielded six spots from pI 5.7 to 7.2 when analysed by two-dimensional electrophoresis; in contrast, MP50 gave one main spot (corresponding to spot 2 of liver GDH) with a pI of approx. 6.5. Soluble liver GDH from commercial sources exhibited a very low or no microtubule-binding activity. In conclusion, we have found a membrane-bound form of GDH capable of specific and nucleotide-sensitive interaction with microtubules. Our data suggest that GDH isoproteins, the number of which has been undervalued up to now, could have cellular functions other than that of an enzyme. Images Figure 1 Figure 3

  9. Cryptotanshinone Suppresses Androgen Receptor-mediated Growth in Androgen Dependent and Castration Resistant Prostate Cancer Cells

    PubMed Central

    Xu, Defeng; Lin, Tzu-Hua; Li, Shaoshun; Da, Jun; Wen, Xing-Qiao; Ding, Jiang; Chang, Chawnshang; Yeh, Shuyuan

    2012-01-01

    Androgen receptor (AR) is the major therapeutic target for the treatment of prostate cancer (PCa). Anti-androgens to reduce or prevent androgens binding to AR are widely used to suppress AR-mediated PCa growth; however, the androgen depletion therapy is only effective for a period of time. Here we found a natural product/Chinese herbal medicine cryptotanshinone (CTS), with a structure similar to dihydrotestosterone (DHT), can effectively inhibit the DHT-induced AR transactivation and prostate cancer cell growth. Our results indicated that 0.5 µM CTS effectively suppresses the growth of AR-positive PCa cells, but has little effect on AR negative PC-3 cells and non-malignant prostate epithelial cells. Furthermore, our data indicated that CTS could modulate AR transactivation and suppress the DHT-mediated AR target genes (PSA, TMPRSS2, and TMEPA1) expression in both androgen responsive PCa LNCaP cells and castration resistant CWR22rv1 cells. Importantly, CTS selective inhibits AR without repressing the activities of other nuclear receptors, including ERα, GR, and PR. The mechanistic studies indicate that CTS functions as an AR inhibitor to suppress androgen/AR-mediated cell growth and PSA expression by blocking AR dimerization and the AR–coregulator complex formation. Furthermore, we showed that CTS effectively inhibits CWR22Rv1 cell growth in the xenograft animal model. The previously un-described mechanisms of CTS may explain how CTS inhibits the growth of PCa cells and help us to establish new therapeutic concepts for the treatment of PCa. PMID:22154085

  10. Specific binding sites for inositol 1,3,4,5-tetrakisphosphate are located predominantly in the plasma membranes of human platelets.

    PubMed Central

    Cullen, P J; Patel, Y; Kakkar, V V; Irvine, R F; Authi, K S

    1994-01-01

    In the present study we describe the characterization and localization of Ins(1,3,4,5)P4-binding sites in human platelet membranes. Specific binding sites for Ins(1,3,4,5)P4 have been identified on mixed, plasma and intracellular membranes from neuraminidase-treated platelets using highly purified carrier-free [32P]Ins(1,3,4,5)P4. The displacement of Ins(1,3,4,5)P4 from these sites by Ins(1,4,5)P3 and InsP6 occurs at greater than two orders of magnitude higher concentrations and with Ins(1,3,4,5,6)P5 at about 40-fold higher concentrations than with Ins(1,3,4,5)P4. The membranes were further separated by free-flow electrophoresis into plasma and intracellular membranes. The Ins(1,3,4,5)P4-binding sites separated with plasma membranes, and showed similar affinities and specificities as mixed membranes, whereas Ins(1,4,5)P3-binding sites were predominantly in the intracellular membranes. These results suggest a predominantly plasma membrane location for putative Ins(1,3,4,5)P4 receptors in human platelets. PMID:8141791

  11. Androgen Receptor Repression of GnRH Gene Transcription

    PubMed Central

    Brayman, Melissa J.; Pepa, Patricia A.; Berdy, Sara E.

    2012-01-01

    Alterations in androgen levels lead to reproductive defects in both males and females, including hypogonadotropic hypogonadism, anovulation, and infertility. Androgens have been shown to down-regulate GnRH mRNA levels through an androgen receptor (AR)-dependent mechanism. Here, we investigate how androgen regulates expression from the GnRH regulatory region in the GT1-7 cell line, a model of GnRH neurons. A synthetic androgen, R1881, repressed transcription from the GnRH promoter (GnRH-P) in an AR-dependent manner, and liganded AR associated with the chromatin at the GnRH-P in live GT1-7 cells. The three known octamer-binding transcription factor-1 (Oct-1) binding sites in GnRH-P were required for AR-mediated repression, although other sequences were also involved. Although a multimer of the consensus Oct-1 binding site was not repressed, a multimer of the cluster of Oct-1, Pre-B cell leukemia transcription factor (Pbx)/Prep, and NK2 homeobox 1 (Nkx2.1) binding sites, found at −106/−91 in GnRH-P, was sufficient for repression. In fact, overexpression of any of these factors disrupted the androgen response, indicating that a balance of factors in this tripartite complex is required for AR repression. AR bound to this region in EMSA, indicating a direct interaction of AR with DNA or with other transcription factors bound to GnRH-P at this sequence. Collectively, our data demonstrate that GnRH transcription is repressed by AR via multiple sequences in GnRH-P, including three Oct-1 binding sites, and that this repression requires the complex interaction of several transcription factors. PMID:22074952

  12. A Remote Arene-Binding Site on Prostate Specific Membrane Antigen Revealed by Antibody-Recruiting Small Molecules

    SciTech Connect

    Zhang, Andrew X.; Murelli, Ryan P.; Barinka, Cyril; Michel, Julien; Cocleaza, Alexandra; Jorgensen, William L.; Lubkowski, Jacek; Spiegel, David A.

    2010-09-27

    Prostate specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase overexpressed in many forms of prostate cancer. Our laboratory has recently disclosed a class of small molecules, called ARM-Ps (antibody-recruiting molecule targeting prostate cancer) that are capable of enhancing antibody-mediated immune recognition of prostate cancer cells. Interestingly, during the course of these studies, we found ARM-Ps to exhibit extraordinarily high potencies toward PSMA, compared to previously reported inhibitors. Here, we report in-depth biochemical, crystallographic, and computational investigations which elucidate the origin of the observed affinity enhancement. These studies reveal a previously unreported arene-binding site on PSMA, which we believe participates in an aromatic stacking interaction with ARMs. Although this site is composed of only a few amino acid residues, it drastically enhances small molecule binding affinity. These results provide critical insights into the design of PSMA-targeted small molecules for prostate cancer diagnosis and treatment; more broadly, the presence of similar arene-binding sites throughout the proteome could prove widely enabling in the optimization of small molecule-protein interactions.

  13. Dietary intake of sodium chloride in the rat influences [3H]nitrendipine binding to adrenal glomerulosa cell membranes but does not alter binding to vascular smooth muscle membranes.

    PubMed Central

    Schiebinger, R J

    1985-01-01

    Angiotensin II-stimulated secretion by adrenal glomerulosa cells and contraction by vascular smooth muscle (VSM) are dependent on calcium influx through membrane calcium channels. We have examined the hypothesis that the altered responsiveness of adrenal glomerulosa cells and VSM to angiotensin II during NaCl restriction may be associated with a change in membrane calcium channel number. To test this hypothesis, female rats were placed on a high or low NaCl diet. On the 14th day, membranes were prepared from the zona glomerulosa, aorta, mesenteric artery, and uterus. [3H]Nitrendipine binding was used to monitor calcium channel number. The [3H]nitrendipine binding capacity was observed to be higher in the zona glomerulosa during NaCl restriction than during high NaCl intake (83 +/- 18 vs. 49 +/- 9 fmol/mg protein, P less than 0.025, n = 6 paired experiments). The binding capacities of [3H]nitrendipine on the low and high NaCl diet were similar in the mesenteric artery (10 +/- 1 vs. 9 +/- 1 fmol/mg protein, n = 8), aorta (33 +/- 5 vs. 35 +/- 8 fmol/mg protein, n = 5), or uterus (87 +/- 15 vs. 85 +/- 16 fmol/mg protein, n = 4), respectively. The dissociation constants of [3H]nitrendipine binding did not differ on a low or high NaCl intake in the zona glomerulosa (0.84 +/- .12 vs. 0.79 +/- .10 nM), mesenteric artery (0.82 +/- .06 vs. 83 +/- .05 nM), aorta (0.90 +/- .11 vs. 0.92 +/- .12 nM), or uterus (0.55 +/- .12 vs. 0.56 +/- .10 nM), respectively. We conclude that the blunted response of VSM to angiotensin II during NaCl restriction is best explained by the previously reported lower number of angiotensin II receptors since calcium channel number does not change. In the adrenal glomerulosa cell, NaCl restriction is associated with a higher number of membrane calcium channels and angiotensin II receptors. The increase in calcium channel number may reflect the influence of an unknown factor(s) believed to be necessary for the full expression of the adrenal glomerulosa

  14. Prolactin/Stat5 and androgen R1881 coactivate carboxypeptidase-D gene in breast cancer cells.

    PubMed

    Koirala, Samir; Thomas, Lynn N; Too, Catherine K L

    2014-03-01

    Plasma membrane-bound carboxypeptidase-D (CPD) cleaves C-terminal arginine from extracellular substrates. In the cell, arginine is converted to nitric oxide (NO). We have reported that up-regulation of CPD mRNA/protein levels by 17β-estradiol and prolactin (PRL) in breast cancer cells, and by testosterone in prostate cancer cells, increased NO production and cell survival. The CPD promoter contains a consensus γ-interferon-activated sequence (GAS) and 3 putative androgen response elements (ARE.1, ARE.2, ARE.3) that could potentially bind PRL-activated transcription factor Stat5 (signal transducer and activator of transcription 5) and the liganded androgen receptor (AR), respectively. This study showed that synthetic androgen R1881 and PRL elevated CPD mRNA/protein levels in human MCF-7 and T47D breast cancer cells in a time-/dose-dependent manner. PRL/R1881-elevated CPD expression was blocked by actinomycin-D, and a CPD promoter construct containing these GAS and AREs was stimulated by PRL or R1881, indicating transcriptional regulation by both hormones. Luciferase reporter assays showed that GAS and the adjacent ARE.1 only were active. Mutation of GAS in the ΔGAS-CPD construct (ARE.1 intact) abolished CPD promoter activity in response to PRL and, surprisingly, to R1881 as well. ΔGAS-CPD promoter activity was restored by PRL+R1881 in combination, and enhanced by ectopic Stat5, but abolished by Stat5 gene knockdown. Chromatin immunoprecipitation analysis confirmed binding of activated Stat5 and liganded AR to GAS and ARE.1, respectively. Activated Stat5 also induced binding of unliganded AR to ARE.1, and liganded AR induced binding of unactivated Stat5 to GAS. In summary, PRL and R1881, acting through Stat5 and AR, act cooperatively to stimulate CPD gene transcription in breast cancer cells. PMID:24433040

  15. Inhibition of 3H-nitrendipine binding in rat aortic and cerebral cortex membranes by the new dihydropyridine calcium antagonist benidipine hydrochloride.

    PubMed

    Ishii, A

    1989-12-01

    Effects of a new calcium antagonist, benidipine hydrochloride (+/-)-(R*)-3-[(R*)-1-benzyl-3-piperidyl]methyl 1,4-dihydro-2,6-dimethyl-4-(m-nitrophenyl)-3,5-pyridinedi carboxylate hydrochloride, KW-3049), on the 3H-nitrendipine binding in the rat aortic microsomes and cerebral cortex membranes were examined. The equilibrium dissociation constant (Kd) and the binding capacity (Bmax) were 0.32 nmol/l and 59.0 fmol/mg protein, respectively, in the rat aorta and 0.17 nmol/l and 450 fmol/mg protein, respectively, in the rat cortex. In both types of membranes, the specific binding was inhibited completely and concentration-dependently by six calcium antagonists such as benidipine, nicardipine, nisoldipine, nitrendipine, nifedipine and flunarizine. Diltiazem enhanced concentration-dependently the 3H-nitrendipine binding in the aortic and cortex membranes. Benidipine had the highest affinity for specific 3H-nitrendipine binding sites in the aorta (Ki = 0.063 nmol/l) and in the cortex membranes (0.043 nmol/l). The decreasing order of binding affinity of the isomers of benidipine for 3H-nitrendipine binding sites was S-S, benidipine, S-R, R-R and R-S. Presumed metabolites had a weak affinity for 3H-nitrendipine binding sites. PMID:2624603

  16. α-Tocopherols modify the membrane dipole potential leading to modulation of ligand binding by P-glycoprotein

    PubMed Central

    Davis, Sterenn; Davis, Benjamin M.; Richens, Joanna L.; Vere, Kelly-Ann; Petrov, Peter G.; Winlove, C. Peter; O’Shea, Paul

    2015-01-01

    α-Tocopherol (vitamin E) has attracted considerable attention as a potential protective or palliative agent. In vitro, its free radical-scavenging antioxidant action has been widely demonstrated. In vivo, however, vitamin E treatment exhibits negligible benefits against oxidative stress. α-Tocopherol influences lipid ordering within biological membranes and its derivatives have been suggested to inhibit the multi-drug efflux pump, P-glycoprotein (P-gp). This study employs the fluorescent membrane probe, 1-(3-sulfonatopropyl)-4-[β[2-(di-n-octylamino)-6-naphthyl]vinyl] pyridinium betaine, to investigate whether these effects are connected via influences on the membrane dipole potential (MDP), an intrinsic property of biological membranes previously demonstrated to modulate P-gp activity. α-Tocopherol and its non-free radical-scavenging succinate analog induced similar decreases in the MDP of phosphatidylcholine vesicles. α-Tocopherol succinate also reduced the MDP of T-lymphocytes, subsequently decreasing the binding affinity of saquinavir for P-gp. Additionally, α-tocopherol succinate demonstrated a preference for cholesterol-treated (membrane microdomain enriched) cells over membrane cholesterol-depleted cells. Microdomain disruption via cholesterol depletion decreased saquinavir’s affinity for P-gp, potentially implicating these structures in the influence of α-tocopherol succinate on P-gp. This study provides evidence of a microdomain dipole potential-dependent mechanism by which α-tocopherol analogs influence P-gp activity. These findings have implications for the use of α-tocopherol derivatives for drug delivery across biological barriers. PMID:26026069

  17. α-Tocopherols modify the membrane dipole potential leading to modulation of ligand binding by P-glycoprotein.

    PubMed

    Davis, Sterenn; Davis, Benjamin M; Richens, Joanna L; Vere, Kelly-Ann; Petrov, Peter G; Winlove, C Peter; O'Shea, Paul

    2015-08-01

    α-Tocopherol (vitamin E) has attracted considerable attention as a potential protective or palliative agent. In vitro, its free radical-scavenging antioxidant action has been widely demonstrated. In vivo, however, vitamin E treatment exhibits negligible benefits against oxidative stress. α-Tocopherol influences lipid ordering within biological membranes and its derivatives have been suggested to inhibit the multi-drug efflux pump, P-glycoprotein (P-gp). This study employs the fluorescent membrane probe, 1-(3-sulfonatopropyl)-4-[β[2-(di-n-octylamino)-6-naphthyl]vinyl] pyridinium betaine, to investigate whether these effects are connected via influences on the membrane dipole potential (MDP), an intrinsic property of biological membranes previously demonstrated to modulate P-gp activity. α-Tocopherol and its non-free radical-scavenging succinate analog induced similar decreases in the MDP of phosphatidylcholine vesicles. α-Tocopherol succinate also reduced the MDP of T-lymphocytes, subsequently decreasing the binding affinity of saquinavir for P-gp. Additionally, α-tocopherol succinate demonstrated a preference for cholesterol-treated (membrane microdomain enriched) cells over membrane cholesterol-depleted cells. Microdomain disruption via cholesterol depletion decreased saquinavir's affinity for P-gp, potentially implicating these structures in the influence of α-tocopherol succinate on P-gp. This study provides evidence of a microdomain dipole potential-dependent mechanism by which α-tocopherol analogs influence P-gp activity. These findings have implications for the use of α-tocopherol derivatives for drug delivery across biological barriers. PMID:26026069

  18. Bombyx mori Midgut Membrane Protein P252, Which Binds to Bacillus thuringiensis Cry1A, Is a Chlorophyllide-Binding Protein, and the Resulting Complex Has Antimicrobial Activity▿

    PubMed Central

    Pandian, Ganesh N.; Ishikawa, Toshiki; Togashi, Makoto; Shitomi, Yasuyuki; Haginoya, Kohsuke; Yamamoto, Shuhei; Nishiumi, Tadayuki; Hori, Hidetaka

    2008-01-01

    The epithelial cell membrane 252-kDa protein (P252) isolated in our laboratory from Bombyx mori midgut was shown to bind strongly with Cry1Aa, Cry1Ab, and Cry1Ac toxins of Bacillus thuringiensis (15). In the current paper, P252 was shown to bind with chlorophyllide (Chlide) to form red fluorescent protein (RFP) complex, termed Bm252RFP, with absorbance and fluorescence emission peaks at 600 nm and 620 nm, respectively. P252 at a concentration of 1 μM is shown to bind with about 50 μM Chlide in a positively cooperative reaction to form Bm252RFP under aerobic conditions and in the presence of light at 37°C. Various parameters influencing this reaction have been optimized for efficient in vitro chemical synthesis of Bm252RFP. Circular dichroism spectra revealed that P252 is composed of a β-structure (39.8% ± 2.2%, based on 5 samples) with negligible contribution of α-helix structure. When bound to Chlide, the β-structure content in the complex is reduced to 21.6% ± 3.1% (n = 5). Since Chlide had no secondary structure, the observed reduction suggests significant conformational changes of P252 during the formation of Bm252RFP complex. Bm252RFP had antimicrobial activity against Escherichia coli, Serratia marcescens, B. thuringiensis, and Saccharomyces cerevisiae with 50% effective concentrations of 2.82, 2.94, 5.88 μM, and 21.6 μM, respectively. This is the first report ever to show clear, concrete binding characteristics of the midgut protein to form an RFP having significant antimicrobial activity. PMID:18192432

  19. Androgen receptor polymorphism (CAG repeats) and androgenicity.

    PubMed

    Canale, D; Caglieresi, C; Moschini, C; Liberati, C D; Macchia, E; Pinchera, A; Martino, E

    2005-09-01

    Objective Polymorphism of the androgen receptor (AR) has been related to various pathophysiological conditions, such as osteoporosis and infertility. The objectives of this study were to evaluate the frequency of distribution in a normal Italian population and to assess CAG repeats (CAGr) in other conditions, such as hypoandrogenism, potentially influenced by AR polymorphism. Patients and measurements CAGr polymorphism was determined in a group of 91 healthy normoandrogenized subjects, 29 hypoandrogenized patients (hypoplasia of prostate and seminal vesicles, reduced beard or body hair, etc.) and 29 infertile patients by direct sequencing. Results The mean (+/- SD) number of CAG repeats [(CAGr)n] was 21.5 (+/- 1.7) in the control group, 21.4 (+/- 2.0) in the infertile patients and 24.0 (+/- 2.9) in the hypoandrogenic males. The difference was statistically significant between this last group and the other two (P < 0.0001), while there was no difference between normal controls and infertile patients. The frequency distribution showed a shift towards higher CAG length in hypoandrogenized patients compared to controls and infertile patients. If we used a cut-off point of 24.9 (2 SD above the mean), the percentage of patients with 25 or more CAGr repeats was 38% among hypoandrogenized patients, 7% among infertile patients and 5% among the control group. In hypoandrogenized subjects (CAGr)n correlated slightly with testis and prostate volume. The number of CAG repeats was not associated with any of the hormonal parameters, including testosterone, evaluated in the three groups. Conclusions Our normal population, representing subjects from Central Italy, is superimposable on other European populations with regard to (CAGr)n distribution. Hypoandrogenic males have a shift in the frequency distribution towards longer (CAGr)n. Infertile patients are not statistically different from the control group. These findings suggest that, given the same amount of circulating

  20. Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes

    PubMed Central

    1978-01-01

    Rat liver rough microsomes (RM) contain two integral membrane proteins which are not found in smooth microsomes (SM) and appear to be related to the presence of ribosome-binding sites. These proteins, of molecular weight 65,000 and 63,000, were designated ribophorins I and II, respectively. They were not released from the microsomal membranes by alkali or acid treatment, or when the ribosomes were detached by incubation with puromycin in a high salt medium. The anionic detergent sodium deoxycholate caused solubilization of the ribophorins, but neutral detergents led to their recovery with the sedimentable ribosomes. Ribosomal aggregates containing both ribophorins, but few other membrane proteins, were obtained from RM treated with the nonionic detergent Kyro EOB (2.5 X10(-2) M) in a low ionic strength medium. Sedimentation patterns produced by these aggregates resembled those of large polysomes but were not affected by RNase treatment. The aggregates, however, were dispersed by mild trypsinization (10 microgram trypsin for 30 min at 0 degrees C), incubation with deoxycholate, or in a medium of high salt concentration. These treatments led to a concomitant degradation or release of the ribophorins. It was estimated, from the staining intensity of protein bands in acrylamide gels, that in the Kyro EOB aggregates there were one to two molecules of each ribophorin per ribosome. Sedimentable complexes without ribosomes containing both ribophorins could also be obtained by dissolving RM previously stripped of ribosomes by puromycin- KCl using cholate, a milder detergent than DOC. Electron microscope examination of the residue obtained from RM treated with Kyro EOB showed that the rapidly sedimenting polysome-like aggregates containing the ribophorins consisted of groups of tightly packed ribosomes which were associated with remnants of the microsomal membranes. PMID:649658

  1. Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes.

    PubMed

    Kreibich, G; Ulrich, B L; Sabatini, D D

    1978-05-01

    Rat liver rough microsomes (RM) contain two integral membrane proteins which are not found in smooth microsomes (SM) and appear to be related to the presence of ribosome-binding sites. These proteins, of molecular weight 65,000 and 63,000, were designated ribophorins I and II, respectively. They were not released from the microsomal membranes by alkali or acid treatment, or when the ribosomes were detached by incubation with puromycin in a high salt medium. The anionic detergent sodium deoxycholate caused solubilization of the ribophorins, but neutral detergents led to their recovery with the sedimentable ribosomes. Ribosomal aggregates containing both ribophorins, but few other membrane proteins, were obtained from RM treated with the nonionic detergent Kyro EOB (2.5 X10(-2) M) in a low ionic strength medium. Sedimentation patterns produced by these aggregates resembled those of large polysomes but were not affected by RNase treatment. The aggregates, however, were dispersed by mild trypsinization (10 microgram trypsin for 30 min at 0 degrees C), incubation with deoxycholate, or in a medium of high salt concentration. These treatments led to a concomitant degradation or release of the ribophorins. It was estimated, from the staining intensity of protein bands in acrylamide gels, that in the Kyro EOB aggregates there were one to two molecules of each ribophorin per ribosome. Sedimentable complexes without ribosomes containing both ribophorins could also be obtained by dissolving RM previously stripped of ribosomes by puromycin-KCl using cholate, a milder detergent than DOC. Electron microscope examination of the residue obtained from RM treated with Kyro EOB showed that the rapidly sedimenting polysome-like aggregates containing the ribophorins consisted of groups of tightly packed ribosomes which were associated with remnants of the microsomal membranes. PMID:649658