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Sample records for membrane androgen binding

  1. Antagonizing effects of membrane-acting androgens on the eicosanoid receptor OXER1 in prostate cancer

    PubMed Central

    Kalyvianaki, Konstantina; Gebhart, Veronika; Peroulis, Nikolaos; Panagiotopoulou, Christina; Kiagiadaki, Fotini; Pediaditakis, Iosif; Aivaliotis, Michalis; Moustou, Eleni; Tzardi, Maria; Notas, George; Castanas, Elias; Kampa, Marilena

    2017-01-01

    Accumulating evidence during the last decades revealed that androgen can exert membrane initiated actions that involve signaling via specific kinases and the modulation of significant cellular processes, important for prostate cancer cell growth and metastasis. Results of the present work clearly show that androgens can specifically act at the membrane level via the GPCR oxoeicosanoid receptor 1 (OXER1) in prostate cancer cells. In fact, OXER1 expression parallels that of membrane androgen binding in prostate cancer cell lines and tumor specimens, while in silico docking simulation of OXER1 showed that testosterone could bind to OXER1 within the same grove as 5-OxoETE, the natural ligand of OXER1. Interestingly, testosterone antagonizes the effects of 5-oxoETE on specific signaling pathways and rapid effects such as actin cytoskeleton reorganization that ultimately can modulate cell migration and metastasis. These findings verify that membrane-acting androgens exert specific effects through an antagonistic interaction with OXER1. Additionally, this interaction between androgen and OXER1, which is an arachidonic acid metabolite receptor expressed in prostate cancer, provides a novel link between steroid and lipid actions and renders OXER1 as new player in the disease. These findings should be taken into account in the design of novel therapeutic approaches in prostate cancer. PMID:28290516

  2. Structural characteristics of anabolic androgenic steroids contributing to binding to the androgen receptor and to their anabolic and androgenic activities. Applied modifications in the steroidal structure.

    PubMed

    Fragkaki, A G; Angelis, Y S; Koupparis, M; Tsantili-Kakoulidou, A; Kokotos, G; Georgakopoulos, C

    2009-02-01

    Anabolic androgenic steroids (AAS) are synthetic derivatives of testosterone introduced for therapeutic purposes providing enhanced anabolic potency with reduced androgenic effects. Androgens mediate their action through their binding to the androgen receptor (AR) which is mainly expressed in androgen target tissues, such as the prostate, skeletal muscle, liver and central nervous system. This paper reviews some of the wide spectrum of testosterone and synthetic AAS structure modifications related to the intended enhancement in anabolic activity. The structural features of steroids necessary for effective binding to the AR and those which contribute to the stipulation of the androgenic and anabolic activities are also presented.

  3. The Stress Response Mediator ATF3 Represses Androgen Signaling by Binding the Androgen Receptor

    PubMed Central

    Wang, Hongbo; Jiang, Ming; Cui, Hongmei; Chen, Mengqian; Buttyan, Ralph; Hayward, Simon W.; Hai, Tsonwin; Wang, Zhengxin

    2012-01-01

    Activating transcription factor 3 (ATF3) is a common mediator of cellular stress response signaling and is often aberrantly expressed in prostate cancer. We report here that ATF3 can directly bind the androgen receptor (AR) and consequently repress AR-mediated gene expression. The ATF3-AR interaction requires the leucine zipper domain of ATF3 that independently binds the DNA-binding and ligand-binding domains of AR, and the interaction prevents AR from binding to cis-acting elements required for expression of androgen-dependent genes while inhibiting the AR N- and C-terminal interaction. The functional consequences of the loss of ATF3 expression include increased transcription of androgen-dependent genes in prostate cancer cells that correlates with increased ability to grow in low-androgen-containing medium and increased proliferative activity of the prostate epithelium in ATF3 knockout mice that is associated with prostatic hyperplasia. Our results thus demonstrate that ATF3 is a novel repressor of androgen signaling that can inhibit AR functions, allowing prostate cells to restore homeostasis and maintain integrity in the face of a broad spectrum of intrinsic and environmental insults. PMID:22665497

  4. 9S binding protein for androgens and progesterone.

    PubMed

    Wilson, E M; Lea, O A; French, F S

    1977-05-01

    A steroid binding protein fraction with a sedimentation coefficient of approximately 9 S (molecular weight approximately equal to 200,000) has been identified in 105,000 X g supernatants of several androgen-responsive organs. Highest concentrations were found in epididymis and testis, but small amounts were detected in prostate, seminal vesicle, kidney, submandibular gland, and lung. The 9S protein binds [3H]dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) and [3H]progesterone (4-pregnene-3,20-dione) with equilibrium binding constants of approximately 10(5) M-1 and 10(6) M-1, respectively. The concentration of 9S binding sites in epididymis is approximately 10(-11) mol/mg of supernatant protein, which is at least 10(5) times greater than the concentration of androgen receptor. 9S binding protein appears to be a nonsecretory, intracellular protein and has properties different from the andorgen receptor. It is unretarded on DEAE-Sephadex chromatography at pH 8.0, and its sedimentation rate on sucrose gradients is not altered at high ionic strength (0.4 M KCl). Like the androgen receptor, its binding activity, which is maximal between pH 7 and 9.5, is heat labile, decreased by sulfhydryl reagents, and enhanced by 2-mercaptoethanol. It is suggested that because of its high concentration and low affinity, 9S binding protein may function in the intracellular accumulation of compartmentalization of androgens or progesterone.

  5. Discordant measures of androgen-binding kinetics in two mutant androgen receptors causing mild or partial androgen insensitivity, respectively.

    PubMed

    Shkolny, D L; Beitel, L K; Ginsberg, J; Pekeles, G; Arbour, L; Pinsky, L; Trifiro, M A

    1999-02-01

    We have characterized two different mutations of the human androgen receptor (hAR) found in two unrelated subjects with androgen insensitivity syndrome (AIS): in one, the external genitalia were ambiguous (partial, PAIS); in the other, they were male, but small (mild, MAIS). Single base substitutions have been found in both individuals: E772A in the PAIS subject, and R871G in the MAIS patient. In COS-1 cells transfected with the E772A and R871G hARs, the apparent equilibrium dissociation constants (Kd) for mibolerone (MB) and methyltrienolone are normal. Nonetheless, the mutant hAR from the PAIS subject (E772A) has elevated nonequilibrium dissociation rate constants (k(diss)) for both androgens. In contrast, the MAIS subject's hAR (R871G) has k(diss) values that are apparently normal for MB and methyltrienolone; in addition, the R871G hAR's ability to bind MB resists thermal stress better than the hAR from the PAIS subject. The E772A and R871G hARs, therefore, confer the same pattern of discordant androgen-binding parameters in transfected COS-1 cells as observed previously in the subjects' genital skin fibroblasts. This proves their pathogenicity and correlates with the relative severity of the clinical phenotype. In COS-1 cells transfected with an androgen-responsive reporter gene, trans-activation was 50% of normal in cells containing either mutant hAR. However, mutant hAR-MB binding is unstable during prolonged incubation with MB, whereas normal hAR-MB binding increases. Thus, normal equilibrium dissociation constants alone, as determined by Scatchard analysis, may not be indicative of normal hAR function. An increased k(diss) despite a normal Kd for a given androgen suggests that it not only has increased egress from a mutant ligand-binding pocket, but also increased access to it. This hypothesis has certain implications in terms of the three-dimensional model of the ligand-binding domain of the nuclear receptor superfamily.

  6. 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 ...

  7. 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...

  8. A comparison of progestin and androgen receptor binding using the CoMFA technique

    NASA Astrophysics Data System (ADS)

    Loughney, Deborah A.; Schwender, Charles F.

    1992-12-01

    A series of 48 steroids has been studied with the SYBYL QSAR module using Relative Binding Affinities (RBAs) to progesterone and androgen receptors obtained from the literature. Models for the progesterone and androgen data were developed. Both models show regions where sterics and electrostatics correlate to binding affinity but are different for androgen and progesterone which suggests differences possibly important for receptor selectivity. The progesterone model is more predictive than the androgen (predictive r2 of 0.725 vs. 0.545 for progesterone and androgen, respectively).

  9. Loss of androgen receptor binding to selective androgen response elements causes a reproductive phenotype in a knockin mouse model

    PubMed Central

    Schauwaers, Kris; De Gendt, Karel; Saunders, Philippa T. K.; Atanassova, Nina; Haelens, Annemie; Callewaert, Leen; Moehren, Udo; Swinnen, Johannes V.; Verhoeven, Guido; Verrijdt, Guy; Claessens, Frank

    2007-01-01

    Androgens influence transcription of their target genes through the activation of the androgen receptor (AR) that subsequently interacts with specific DNA motifs in these genes. These DNA motifs, called androgen response elements (AREs), can be classified in two classes: the classical AREs, which are also recognized by the other steroid hormone receptors; and the AR-selective AREs, which display selectivity for the AR. For in vitro interaction with the selective AREs, the androgen receptor DNA-binding domain is dependent on specific residues in its second zinc-finger. To evaluate the physiological relevance of these selective elements, we generated a germ-line knockin mouse model, termed SPARKI (SPecificity-affecting AR KnockIn), in which the second zinc-finger of the AR was replaced with that of the glucocorticoid receptor, resulting in a chimeric protein that retains its ability to bind classical AREs but is unable to bind selective AREs. The reproductive organs of SPARKI males are smaller compared with wild-type animals, and they are also subfertile. Intriguingly, however, they do not display any anabolic phenotype. The expression of two testis-specific, androgen-responsive genes is differentially affected by the SPARKI mutation, which is correlated with the involvement of different types of response elements in their androgen responsiveness. In this report, we present the first in vivo evidence of the existence of two functionally different types of AREs and demonstrate that AR-regulated gene expression can be targeted based on this distinction. PMID:17360365

  10. Structure of the homodimeric androgen receptor ligand-binding domain

    PubMed Central

    Nadal, Marta; Prekovic, Stefan; Gallastegui, Nerea; Helsen, Christine; Abella, Montserrat; Zielinska, Karolina; Gay, Marina; Vilaseca, Marta; Taulès, Marta; Houtsmuller, Adriaan B.; van Royen, Martin E.; Claessens, Frank; Fuentes-Prior, Pablo; Estébanez-Perpiñá, Eva

    2017-01-01

    The androgen receptor (AR) plays a crucial role in normal physiology, development and metabolism as well as in the aetiology and treatment of diverse pathologies such as androgen insensitivity syndromes (AIS), male infertility and prostate cancer (PCa). Here we show that dimerization of AR ligand-binding domain (LBD) is induced by receptor agonists but not by antagonists. The 2.15-Å crystal structure of homodimeric, agonist- and coactivator peptide-bound AR-LBD unveils a 1,000-Å2 large dimerization surface, which harbours over 40 previously unexplained AIS- and PCa-associated point mutations. An AIS mutation in the self-association interface (P767A) disrupts dimer formation in vivo, and has a detrimental effect on the transactivating properties of full-length AR, despite retained hormone-binding capacity. The conservation of essential residues suggests that the unveiled dimerization mechanism might be shared by other nuclear receptors. Our work defines AR-LBD homodimerization as an essential step in the proper functioning of this important transcription factor. PMID:28165461

  11. 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...

  12. Androgen-induced sexual dimorphism in high affinity dopamine binding in the brain transcends the hypothalamic-limbic region.

    PubMed Central

    Jalilian-Tehrani, M. H.; Karakiulakis, G.; Le Blond, C. B.; Powell, R.; Thomas, P. J.

    1982-01-01

    1 High affinity binding of [3H]-dopamine and [3H]-5-hydroxytryptamine ([3H]-5-HT) was measured in membrane fractions prepared from cerebral cortex, amygdala, hypothalamus, thalamus and brain stem of rats of either sex and of rats which had been either neonatally castrated or androgenized. 2 Binding was measured in rats of 8, 20 and 30 days old as well as in adults. 3 [3H]-dopamine bound with approximately 30 nM affinity ahd [3H]-5-HT with approximately 10 nM affinity to all areas of the brain tested. The relative inhibitory effects of haloperidol, apomorphine, cis-flupenthixol, unlabelled dopamine, noradrenaline, spiroperone, (+)-butaclamol, fluphenazine, pimozide and 5-HT on [3H]-dopamine binding in the cerebral cortex was consistent with receptor status for the binding components there as were the relative inhibitory effects of methysergide, dopamine, fluoxetine and ouabain on [3H]-5-HT binding in the fore brain. 4 Neither [3H]-dopamine nor [3H]-5-HT binding varied with the state of the sexual cycle in females. 5 There were no sexual differences in [3H]-5-HT binding in any of the brain areas tested nor was it affected by neonatal androgenization or neonatal castration. 6 [3H]-dopamine binding was greater in the cerebral cortex and amygdala of male than of female rats. These differences could be mimicked artificially by neonatal castration of males (female type development) or neonatal androgenization of females (male type development). Sexual dimorphism did not become overt until 20 days of age and did not extend to hypothalamus, thalamus or brain stem. 7 It is concluded that neonatal sex differences in exposure to steroid hormones has permanent effects on the number of dopamine binding sites in the cerebral cortex and is suggested that this sexual dimorphism extends to the amygdala. PMID:7074286

  13. Partial androgen insensitivity and correlations with the predicted three dimensional structure of the androgen receptor ligand-binding domain.

    PubMed

    Yong, E L; Tut, T G; Ghadessy, F J; Prins, G; Ratnam, S S

    1998-02-01

    Genetic defects of the human androgen receptor (AR) can cause a wide spectrum of androgen insensitivity syndromes (AIS) ranging from phenotypic females in those with complete AIS; ambiguous genitalia in partial AIS; to male infertility in minimal AIS. The majority of these defects are due to point mutations resulting in amino acid substitutions. It is however unclear why certain mutations result in partial AIS, whereas others in the same exon cause the complete syndrome. We present a case of partial AIS due to a point mutation affecting codon 758 of the AR ligand-binding domain (LBD) that changed the sense of the codon from asparagine to threonine (N758T). The mutant receptor displayed normal binding affinity to DHT but abnormal dissociation kinetics in both patient's fibroblasts and transfected COS-7 cells. The mutant AR was thermolabile, and resulted in approximately 50% reduction in receptor transactivation capacity when examined with a reporter gene incorporating an androgen-response-element. Although the 3-D structure of AR LBD is not known, the homologous region in a member of the steroid receptor superfamily, retinoid-X receptor (RXR-alpha), has been crystallized, allowing comparison of aligned amino-acid sequences of RXR-alpha and AR. The mutation, N758T, lies in a predicted linker region between the fifth alpha-helix (H5) and the first beta-strand (S1). Generally, mutations leading to partial AIS tend to cluster in the predicted linker regions located between the structural helices of the AR LBD. Most strikingly, the predicted linker regions contain over 70% of the mutant ARs associated with prostate cancer in the LBD. The occurrence of mutations associated with both partial AIS and prostate cancer in the same predicted linker regions, suggest that this clustering is not coincidental and that the predicted linker regions are likely to have important, but subtle, roles in defining androgen binding and ligand specificity.

  14. Optical biosensor analysis in studying new synthesized bicalutamide analogs binding to androgen receptor.

    PubMed

    Fortugno, Cecilia; Varchi, Greta; Guerrini, Andrea; Carrupt, Pierre-Alain; Bertucci, Carlo

    2014-07-01

    Bicalutamide (Casodex®) is a non-steroidal anti-androgen drug used in the treatment of prostate cancer, which represents the second most common malignancy diagnosed in men worldwide. In this work, we analyze the ability of some novel bicalutamide analogs to bind the androgen receptor, by using an optical biosensor. Androgen receptor was covalently immobilized on a carboxy methyl dextran matrix. The immobilized receptor chip was then used for the binding experiments of the bicalutamide analogs. The (R)-bicalutamide dissociation constant was in good agreement to the value reported in literature obtained by using radiolabeled targets. Most of the new synthesized compounds showed higher androgen receptor binding level, when compared to the reference. Our results clearly indicate that the surface plasmon resonance (SPR) technique offers many advantages with respect to other available technologies in terms of studying biomolecular interactions. Moreover, this study provides an effective methodology for determining the binding affinity of novel chemical entities for the isolated androgen receptor, thus excluding possible off-target interactions occurring in conventional cell-based techniques.

  15. Human sex hormone-binding globulin binding affinities of 125 structurally diverse chemicals and comparison with their binding to androgen receptor, estrogen receptor, and α-fetoprotein.

    PubMed

    Hong, Huixiao; Branham, William S; Ng, Hui Wen; Moland, Carrie L; Dial, Stacey L; Fang, Hong; Perkins, Roger; Sheehan, Daniel; Tong, Weida

    2015-02-01

    One endocrine disruption mechanism is through binding to nuclear receptors such as the androgen receptor (AR) and estrogen receptor (ER) in target cells. The concentration of a chemical in serum is important for its entry into the target cells to bind the receptors, which is regulated by the serum proteins. Human sex hormone-binding globulin (SHBG) is the major transport protein in serum that can bind androgens and estrogens and thus change a chemical's availability to enter the target cells. Sequestration of an androgen or estrogen in the serum can alter the chemical elicited AR- and ER-mediated responses. To better understand the chemical-induced endocrine activity, we developed a competitive binding assay using human pregnancy plasma and measured the binding to the human SHBG for 125 structurally diverse chemicals, most of which were known to bind AR and ER. Eighty seven chemicals were able to bind the human SHBG in the assay, whereas 38 chemicals were nonbinders. Binding data for human SHBG are compared with that for rat α-fetoprotein, ER and AR. Knowing the binding profiles between serum and nuclear receptors will improve assessment of a chemical's potential for endocrine disruption. The SHBG binding data reported here represent the largest data set of structurally diverse chemicals tested for human SHBG binding. Utilization of the SHBG binding data with AR and ER binding data could enable better evaluation of endocrine disrupting potential of chemicals through AR- and ER-mediated responses since sequestration in serum could be considered.

  16. Novel mutation in the ligand-binding domain of the androgen receptor gene (l790p) associated with complete androgen insensitivity syndrome.

    PubMed

    Raicu, Florina; Giuliani, Rossella; Gatta, Valentina; Palka, Chiara; Franchi, Paolo Guanciali; Lelli-Chiesa, Pierluigi; Tumini, Stefano; Stuppia, Liborio

    2008-07-01

    Mutations in the X-linked androgen receptor (AR) gene cause androgen insensitivity syndrome (AIS), resulting in an impaired embryonic sex differentiation in 46,XY genetic men. Complete androgen insensitivity (CAIS) produces a female external phenotype, whereas cases with partial androgen insensitivity (PAIS) have various ambiguities of the genitalia. Mild androgen insensitivity (MAIS) is characterized by undermasculinization and gynecomastia. Here we describe a 2-month-old 46,XY female patient, with all of the characteristics of CAIS. Defects in testosterone (T) and dihydrotestosterone (DHT) synthesis were excluded. Sequencing of the AR gene showed the presence in exon 6 of a T to C transition in the second base of codon 790, nucleotide position 2369, causing a novel missense Leu790Pro mutation in the ligand-binding domain of the AR protein. The identification of a novel AR mutation in a girl with CAIS provides significant information due to the importance of missense mutations in the ligand-binding domain of the AR, which are able to induce functional abnormalities in the androgen binding capability, stabilization of active conformation, or interaction with coactivators.

  17. 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.

  18. Differential DNA binding by the androgen and glucocorticoid receptors involves the second Zn-finger and a C-terminal extension of the DNA-binding domains.

    PubMed Central

    Schoenmakers, E; Alen, P; Verrijdt, G; Peeters, B; Verhoeven, G; Rombauts, W; Claessens, F

    1999-01-01

    The androgen and glucocorticoid hormones evoke specific in vivo responses by activating different sets of responsive genes. Although the consensus sequences of the glucocorticoid and androgen response elements are very similar, this in vivo specificity can in some cases be explained by differences in DNA recognition between both receptors. This has clearly been demonstrated for the androgen response element PB-ARE-2 described in the promoter of the rat probasin gene. Swapping of different fragments between the androgen- and glucocorticoid-receptor DNA-binding domains demonstrates that (i) the first Zn-finger module is not involved in this sequence selectivity and (ii) that residues in the second Zn-finger as well as a C-terminal extension of the DNA-binding domain from the androgen receptor are required. For specific and high-affinity binding to response elements, the DNA-binding domains of the androgen and glucocorticoid receptors need a different C-terminal extension. The glucocorticoid receptor requires 12 C-terminal amino acids for high affinity DNA binding, while the androgen receptor only involves four residues. However, for specific recognition of the PB-ARE-2, the androgen receptor also requires 12 C-terminal residues. Our data demonstrate that the mechanism by which the androgen receptor binds selectively to the PB-ARE-2 is different from that used by the glucocorticoid receptor to bind a consensus response element. We would like to suggest that the androgen receptor recognizes response elements as a direct repeat rather than the classical inverted repeat. PMID:10417312

  19. Study of 202 natural, synthetic, and environmental chemicals for binding to the androgen receptor.

    PubMed

    Fang, Hong; Tong, Weida; Branham, William S; Moland, Carrie L; Dial, Stacy L; Hong, Huixiao; Xie, Qian; Perkins, Roger; Owens, William; Sheehan, Daniel M

    2003-10-01

    A number of environmental and industrial chemicals are reported to possess androgenic or antiandrogenic activities. These androgenic endocrine disrupting chemicals may disrupt the endocrine system of humans and wildlife by mimicking or antagonizing the functions of natural hormones. The present study developed a low cost recombinant androgen receptor (AR) competitive binding assay that uses no animals. We validated the assay by comparing the protocols and results from other similar assays, such as the binding assay using prostate cytosol. We tested 202 natural, synthetic, and environmental chemicals that encompass a broad range of structural classes, including steroids, diethylstilbestrol and related chemicals, antiestrogens, flutamide derivatives, bisphenol A derivatives, alkylphenols, parabens, alkyloxyphenols, phthalates, siloxanes, phytoestrogens, DDTs, PCBs, pesticides, organophosphate insecticides, and other chemicals. Some of these chemicals are environmentally persistent and/or commercially important, but their AR binding affinities have not been previously reported. To the best of our knowledge, these results represent the largest and most diverse data set publicly available for chemical binding to the AR. Through a careful structure-activity relationship (SAR) examination of the data set in conjunction with knowledge of the recently reported ligand-AR crystal structures, we are able to define the general structural requirements for chemical binding to AR. Hydrophobic interactions are important for AR binding. The interaction between ligand and AR at the 3- and 17-positions of testosterone and R1881 found in other chemical classes are discussed in depth. The SAR studies of ligand binding characteristics for AR are compared to our previously reported results for estrogen receptor binding.

  20. 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.

  1. Inhibition of androgen receptor binding by natural and synthetic steroids in cultured human genital skin fibroblasts.

    PubMed

    Breiner, M; Romalo, G; Schweikert, H U

    1986-08-15

    The ability of various natural and synthetic steroids (some of which are widely used in clinical practice) to compete with dihydrotestosterone receptor binding in human genital skin fibroblasts was studied. Binding was assessed in fibroblast monolayers after incubation for 1 h at 37 degrees C with 2 nM 3H-dihydrotestosterone in the presence or absence of increasing concentrations of the steroid to be tested. Inhibition constants (Ki) were determined as the concentration of competitor-required for 50% inhibition of 3H-dihydrotestosterone binding. In addition, relative binding activity (RBA) of each test compound was calculated. Each competitor was tested in at least two different cell strains. The concentrations of unlabeled methyltrienolone (a synthetic nonmetabolizable androgen) and dihydrotestosterone for 50% inhibition of 3H-dihydrotestosterone binding were in the same order of magnitude, namely, 2 nM (2.2 respectively, 2.4 nM), whereas the affinity of testosterone was approximately one-fifth that of dihydrotestosterone. Other potent competitors for dihydrotestosterone binding were three progestins (norgestrel, gestoden, and medroxyprogesterone acetate) which have Ki values similar to testosterone. An order of magnitude lower Ki values (around 10(-7) M) were found for the androgen 17 alpha-propylmesterolone, the antiandrogen cyproterone acetate, and the progestin norethisterone acetate. Binding affinities of all other steroids to the androgen receptor were markedly lower and showed the following order of potency: estrogens (estradiol, ethinyl estradiol, diethylstilbestrol) greater than glucocorticoids as well as aromatase inhibitors and potassium canrenoate.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Expression of androgen-binding protein (ABP) in human cardiac myocytes.

    PubMed

    Schock, H W; Herbert, Z; Sigusch, H; Figulla, H R; Jirikowski, G F; Lotze, U

    2006-04-01

    Cardiomyocytes are known to be androgen targets. Changing systemic steroid levels are thought to be linked to various cardiac ailments, including dilated cardiomyopathy (DCM). The mode of action of gonadal steroid hormones on the human heart is unknown to date. In the present study, we used high-resolution immunocytochemistry on semithin sections (1 microm thick), IN SITU hybridization, and mass spectrometry to investigate the expression of androgen-binding protein (ABP) in human myocardial biopsies taken from male patients with DCM. We observed distinct cytoplasmic ABP immunoreactivity in a fraction of the myocytes. IN SITU hybridization with synthetic oligonucleotide probes revealed specific hybridization signals in these cells. A portion of the ABP-positive cells contained immunostaining for androgen receptor. With SELDI TOF mass spectrometry of affinity purified tissue extracts of human myocardium, we confirmed the presence of a 50 kDa protein similar to ABP. Our observations provide evidence of an intrinsic expression of ABP in human heart. ABP may be secreted from myocytes in a paracrine manner perhaps to influence the bioavailabity of gonadal steroids in myocardium.

  3. Androgen regulated expression of a spermine binding protein gene in mouse ventral prostate.

    PubMed Central

    Mills, J S; Needham, M; Parker, M G

    1987-01-01

    A full length cDNA (MP25) encoding the major mouse prostatic secretory glycoprotein (p25), whose expression is androgen dependent, has been cloned and characterised. Steady-state levels of mRNA are decreased approximately 100-fold after 3 days castration but are restored progressively over 4 days with testosterone treatment. The secreted glycoprotein appears to be a spermine binding protein since the nucleotide and predicted amino acid sequence of MP25 shares extensive homology with a spermine binding protein (SBP) found in rat ventral prostate. Genomic clones indicate that there is a single gene for SBP which consists of 4 exons, the first of which is only 11bp in length. The second exon encodes the signal peptide, the third contains a portion of the spermine binding protein unique to the mouse and the largest exon encodes the bulk of the secreted protein. Images PMID:3502715

  4. A naturally occurring mutation in the human androgen receptor of a subject with complete androgen insensitivity confers binding and transactivation by estradiol.

    PubMed

    Bonagura, Thomas W; Deng, Min; Brown, Terry R

    2007-01-15

    The clinical phenotype of complete androgen insensitivity (CAIS) was associated with a mutation in the human androgen receptor (hAR) gene encoding the amino acid substitution, M745I, in the hAR protein. Transcriptional activation of hAR(M745I) by the synthetic androgen, methyltrienolone (R1881), was reduced compared to wild-type (wt) hAR. The transcriptional co-activator, androgen receptor associated protein 70 (ARA70), failed to enhance transactivation of hAR(M745I) at lower concentrations of R1881 (0.01-0.1 nM), whereas the p160 co-activators, SRC-1 and TIF2, stimulated activity. Transcriptional activity of hAR(M745I) was stimulated by 1 or 10 nM R1881 and activity was further enhanced by co-expression of ARA70 similar to that of the hAR(wt). Transcriptional activity of hAR(wt) was minimally stimulated by estradiol (E2) without or with co-expression of ARA70, whereas 10 or 100 nM E2 increased transactivation by hAR(M745I) of the androgen-responsive MMTV-luciferase reporter gene by 10-fold and activity was further enhanced by ARA70. Increasing concentrations of E2 competed more effectively for binding of R1881 to hAR(M745I) than to hAR(wt), indicative of the preferential binding of E2 to the mutant hAR. Partial tryptic digestion of hAR wt and M745I revealed that activation of the mutant protein was reduced in the presence of R1881. By contrast, tryptic digestion showed that the mutant hAR was activated by the binding of E2. In conclusion, the clinical phenotype of CAIS resulted from a hAR gene mutation encoding hAR(M745I) with reduced binding and transactivation by androgens, but the novel properties of enhanced affinity for and increased transactivation by estradiol.

  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. Interaction of bilirubin with human erythrocyte membranes. Bilirubin binding to neuraminidase- and phospholipase-treated membranes.

    PubMed

    Sato, H; Aono, S; Semba, R; Kashiwamata, S

    1987-11-15

    Saturable bilirubin binding to human erythrocyte membranes was measured before and after digestion with neuraminidase and phospholipases. Neuraminidase-treated erythrocyte membranes did not show any change in their binding properties, indicating that gangliosides could be excluded as candidates for saturable bilirubin-binding sites on erythrocyte membranes. Although bilirubin-binding properties of the membranes did not change after phospholipase D digestion, either, phospholipase C treatment greatly enhanced bilirubin binding. Thus it is suggested that a negatively charged phosphoric acid moiety of phospholipids on the membrane surface may play a role to prevent a large amount of bilirubin from binding to the membranes. Further saturable bilirubin binding to inside-out sealed erythrocyte membrane vesicles showed values comparable with those of the right-side-out sealed membranes, suggesting that the bilirubin-binding sites may be distributed on both outer and inner surfaces of the membranes, or may exist in the membranes where bilirubin may be accessible from either side.

  7. Poly (A) Binding Protein Cytoplasmic 1 Is a Novel Co-Regulator of the Androgen Receptor

    PubMed Central

    Eisermann, Kurtis; Dar, Javid A.; Dong, Jun; Wang, Dan; Masoodi, Khalid Z.; Wang, Zhou

    2015-01-01

    The androgen receptor (AR) is a member of the steroid receptor superfamily that regulates gene expression in a ligand-dependent manner. The NTD of the AR plays a key role in AR transactivation including androgen-independent activation of the AR in castration-resistant prostate cancer (CRPC) cells. We recently reported that amino acids (a.a.) 50-250 of the NTD are capable of modulating AR nucleocytoplasmic trafficking. To further explore the mechanism associated with a.a. 50-250, GFP pull-down assays were performed in C4-2 CRPC cells transfected with GFP tagged a.a. 50-250 of the AR. Mass spectrometry analysis of the pulled down proteins identified poly (A) binding protein cytoplasmic 1 (PABPC1) interaction with this region of the AR. In silico analysis of gene expression data revealed PABPC1 up-regulation in prostate cancer tissue specimens and this up-regulation correlates to increased disease recurrence. Co-immunoprecipitation assays confirmed the association of PABPC1 with a.a. 50-250 of the NTD of the AR. Knockdown of PABPC1 decreased nuclear AR protein levels and inhibited androgen activation of the AR target PSA in LNCaP and C4-2 cells. Additionally, knockdown of PABPC1 inhibited transactivation of the PSA promoter by NAR (AR lacking the LBD) and attenuated proliferation of AR-positive prostate cancer cells. These findings suggest that PABPC1 is a novel co-regulator of the AR and may be a potential target for blocking activation of the AR in CRPC. PMID:26176602

  8. Comparison of rabbit androgen binding protein with testosterone estradiol binding globulin--I. Physical and chemical properties.

    PubMed

    Cheng, S L; Kotite, N; Musto, N A

    1984-12-01

    Rabbit epididymal androgen binding protein (rbABP) and serum testosterone estradiol binding globulin (rbTeBG) were purified and their physicochemical properties compared. Both proteins bound dihydrotestosterone (DHT) with high affinity. Both contained two components, Heavy (H) and Light (L), and their molecular weights and pI values were comparable. rbABP and rbTeBG were different with regard to their ConA-Sepharose binding property. rbABP was not bound by ConA-Sepharose while rbTeBG was found and retained by this lectin; thus, rbABP and rbTeBG differed in their carbohydrate structure. Peptide mapping on SDS-PAGE indicated that the H components of rbABP and rbTeBG were distinct even though they showed a high degree of homology. By contrast, the L components of these two proteins appeared to be identical. The structure of the steroid binding sites of these two proteins was analyzed by peptide mapping of [1,2(3)H]17 beta hydroxy-androsta-4,6-dien-3-one photoaffinity labeled protein. The size distribution of radioactive peptide fragments generated appeared to be identical for these two proteins. However, the distribution of labeled peptides was slightly different when examined by high pressure liquid chromatography (HPLC). The observations suggest that the differences between rbABP and rbTeBG might reside not only in carbohydrate moieties but also in their amino acid sequences.

  9. A novel point mutation in the hormone binding domain of the androgen receptor associated with partial and minimal androgen insensitivity syndrome.

    PubMed

    Galli-Tsinopoulou, Assimina; Hiort, Olaf; Schuster, Tobias; Messer, Gerald; Kuhnle, Ursula

    2003-02-01

    Mutations in the coding sequence of the androgen receptor (AR) gene result in a wide range of androgen insensitivity syndromes (AIS). We report an extended family in which at least five male individuals in different generations suffer from partial AIS. The index patient presented at birth with ambiguous genitalia; the karyotype was 46,XY and subsequent sex assignment male. Elevated stimulated testosterone (T) and normal baseline gonadotropins were found. Family history revealed four additional adult males affected with various abnormalities of their external genitalia. Molecular analysis of the coding sequence of the AR gene revealed in all a novel point mutation in exon 6, changing threonine to isoleucine at codon position 800 in the hormone-binding domain. We conclude that phenotypic variations in mild AR defects are striking and can remain undetected even until late in life.

  10. 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

  11. 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...

  12. Dysregulation of sterol response element-binding proteins and downstream effectors in prostate cancer during progression to androgen independence.

    PubMed

    Ettinger, Susan L; Sobel, Richard; Whitmore, Tanis G; Akbari, Majid; Bradley, Dawn R; Gleave, Martin E; Nelson, Colleen C

    2004-03-15

    Androgen ablation, the most common therapeutic treatment used for advanced prostate cancer, triggers the apoptotic regression of prostate tumors. However, remissions are temporary because surviving prostate cancer cells adapt to the androgen-deprived environment and form androgen-independent (AI) tumors. We hypothesize that adaptive responses of surviving tumor cells result from dysregulated gene expression of key cell survival pathways. Therefore, we examined temporal alterations to gene expression profiles in prostate cancer during progression to androgen independence at several time points using the LNCaP xenograft tumor model. Two key genes, sterol response element-binding protein (SREBP)-1 and -2 (SREBP-1a,-1c, and -2), were consistently dysregulated. These genes are known to coordinately control the expression of the groups of enzymes responsible for lipid and cholesterol synthesis. Northern blots revealed modest increased expression of SREBP-1a, -1c, and -2 after castration, and at androgen independence (day 21-28), the expression levels of both SREBP-1a and -1c were significantly greater than precastrate levels. Changes in SREBP-1 and -2 protein expression were observed by Western analysis. SREBP-1 68-kDa protein levels were maintained throughout progression, however, SREBP-2 68-kDa protein expression increased after castration and during progression (3-fold). SREBPs are transcriptional regulators of over 20 functionally related enzymes that coordinately control the metabolic pathways of lipogenesis and cholesterol synthesis, some of which were likewise dysregulated during progression to androgen independence. RNA levels of acyl-CoA-binding protein/diazepam-binding inhibitor and fatty acid synthase decreased significantly after castration, and then, during progression, increased to levels greater than or equal to precastrate levels. Expression of farnesyl diphosphate synthase did not decrease after castration but did increase significantly during

  13. 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

  14. Localization of androgen-binding protein in proliferating Sertoli cells in culture.

    PubMed

    Kierszenbaum, A L; Feldman, M; Lea, O; Spruill, W A; Tres, L L; Petrusz, P; French, F S

    1980-09-01

    The peroxidase and immunofluorescent localization patterns of androgen-binding protein (ABP), a biological marker of Sertoli cell function, have been examined in cultured Sertoli cells isolated from 20- to 22-day-old rats. ABP immunoreactivity in the form of cytoplasmic granules of variable diameter was observed in Sertoli cells with characteristic lipid droplets and a colony-forming, epithelial-like growth pattern. Incubation of cultures with [3H]thymidine demonstrated that Sertoli cells continue to produce ABP while retaining their capability for synthesizing DNA and undergoing mitosis. A variable number of cultured Sertoli cells became morphologically transformed after exposure to follitropin (follicle-stimulating hormone) and pharmacological agents acting on cyclic nucleotide metabolism. The induced change in Sertoli cell shape coincided with a disappearance of ABP-containing granules from the cytoplasm. These observations demonstrate that localization of ABP by immunological techniques is a valuable tool for the characterization of structural and functional properties of Sertoli cell in culture.

  15. Mutations in the ligand-binding domain of the androgen receptor gene cluster in two regions of the gene.

    PubMed

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

    1992-11-01

    We have analyzed the nucleotide sequence of the androgen receptor from 22 unrelated subjects with substitution mutations of the hormone-binding domain. Eleven had the phenotype of complete testicular feminization, four had incomplete testicular feminization, and seven had Reifenstein syndrome. The underlying functional defect in cultured skin fibroblasts included individuals with absent, qualitative, or quantitative defects in ligand binding. 19 of the 21 substitution mutations (90%) cluster in two regions that account for approximately 35% of the hormone-binding domain, namely, between amino acids 726 and 772 and between amino acids 826 and 864. The fact that one of these regions is homologous to a region of the human thyroid hormone receptor (hTR-beta) which is a known cluster site for mutations that cause thyroid hormone resistance implies that this localization of mutations is not a coincidence. These regions of the androgen receptor may be of particular importance for the formation and function of the hormone-receptor complex.

  16. TET2 binds the androgen receptor and loss is associated with prostate cancer.

    PubMed

    Nickerson, M L; Das, S; Im, K M; Turan, S; Berndt, S I; Li, H; Lou, H; Brodie, S A; Billaud, J N; Zhang, T; Bouk, A J; Butcher, D; Wang, Z; Sun, L; Misner, K; Tan, W; Esnakula, A; Esposito, D; Huang, W Y; Hoover, R N; Tucker, M A; Keller, J R; Boland, J; Brown, K; Anderson, S K; Moore, L E; Isaacs, W B; Chanock, S J; Yeager, M; Dean, M; Andresson, T

    2016-11-07

    Genetic alterations associated with prostate cancer (PCa) may be identified by sequencing metastatic tumour genomes to identify molecular markers at this lethal stage of disease. Previously, we characterized somatic alterations in metastatic tumours in the methylcytosine dioxygenase ten-eleven translocation 2 (TET2), which is altered in 5-15% of myeloid, kidney, colon and PCas. Genome-wide association studies previously identified non-coding risk variants associated with PCa and melanoma. We perform fine-mapping of PCa risk across TET2 using genotypes from the PEGASUS case-control cohort and identify six new risk variants in introns 1 and 2. Oligonucleotides containing two risk variants are bound by the transcription factor octamer-binding protein 1 (Oct1/POU2F1) and TET2 and Oct1 expression are positively correlated in prostate tumours. TET2 is expressed in normal prostate tissue and reduced in a subset of tumours from the Cancer Genome Atlas (TCGA). Small interfering RNA-mediated TET2 knockdown (KD) increases LNCaP cell proliferation, migration and wound healing, verifying loss drives a cancer phenotype. Endogenous TET2 bound the androgen receptor (AR) and AR-coactivator proteins in LNCaP cell extracts, and TET2 KD increases prostate-specific antigen (KLK3/PSA) expression. Published data reveal TET2 binding sites and hydroxymethylcytosine proximal to KLK3. A gene co-expression network identified using TCGA prostate tumour RNA-sequencing identifies co-regulated cancer genes associated with 2-oxoglutarate (2-OG) and succinate metabolism, including TET2, lysine demethylase (KDM) KDM6A, BRCA1-associated BAP1, and citric acid cycle enzymes IDH1/2, SDHA/B, and FH. The co-expression signature is conserved across 31 TCGA cancers suggesting a putative role for TET2 as an energy sensor (of 2-OG) that modifies aspects of androgen-AR signalling. Decreased TET2 mRNA expression in TCGA PCa tumours is strongly associated with reduced patient survival, indicating reduced

  17. Steroid binding sites in liver membranes: interplay between glucocorticoids, sex steroids, and pituitary hormones.

    PubMed

    Fernández-Pérez, L; Flores-Morales, A; Chirino-Godoy, R; Díaz-Chico, J C; Díaz-Chico, B N

    2008-04-01

    Steroid hormones activate target cells through specific receptors that discriminate among ligands based upon recognition of distinct structural features. For most known steroids, membrane and nuclear receptors co-exist in many target cells. However, while the structure of the nuclear receptors and their function as transcriptional activators of specific target genes is generally well understood, the identity of the membrane receptors remains elusive. Using pharmacological and biochemical approaches, we are beginning to characterize receptors for glucocorticoids and anabolic-androgenic steroids in male rat liver membranes. Male rat liver endoplasmic reticulum contains two steroid binding sites which are functionally related and associated with a 90-134 kDa oligomeric protein: (1) the low-affinity glucocorticoid binding site (LAGS), composed at least in part of two peptides (37 and 53 kDa) that bind glucocorticoids and (2) the stanozolol binding protein (STBP), composed at least in part of three peptides (22, 31, and 55 kDa) that bind the synthetic androgen stanozolol. These steroid binding proteins have many properties different from those of classical nuclear receptors, with the salient differences being a failure to recognize "classical" ligands for nuclear receptors together with marked differences in biochemical properties and physiological regulation. The mechanism of interaction of glucocorticoids with the LAGS can be clearly distinguished from that with STBP. Moreover, STBP shows an extremely narrow pharmacological profile, being selective for ST and its analog, danazol, among more than 100 steroids and non-steroidal compounds that were assayed, including those that are able to displace glucocorticoids from the LAGS. The level of LAGS activity undergoes dramatic variations following changes from the physiological serum levels of thyroid hormones, glucocorticoids, GH, vitamin A, and E2. However, neither thyroid hormones nor GH have a critical role on STBP

  18. Establishment of a mouse Sertoli cell line producing rat androgen-binding protein (ABP).

    PubMed

    Ducray, A; Bloquel, M; Hess, K; Hammond, G L; Gérard, H; Gérard, A

    1998-01-01

    The ultimate goal of this study was to compare the fate of rat testicular germ cells cocultured with mouse Sertoli cells that either do or do not produce rat androgen-binding protein (ABP). As a first step, we stably transfected a rat ABP expression construct into an immortalized mouse Sertoli cell line (TM4), which does not produce ABP when growing on plastic without hormones. The transfection of the pRc/CMV- rat ABP cDNA expression vector containing a neomycin resistance gene was made by either the liposome method (Dotap) or by polyethyleneimine transfection (PEI) into TM4 cell cultures. Neomycin-resistant clones were selected by adding Geneticin to the culture medium for 3 weeks. Analysis of over 25 clones revealed the presence of recombinant rat ABP when cell extracts and culture media were probed with a rabbit polyclonal antibody raised against rat testicular ABP, indicating the translation and secretion of a protein similar to rat testicular ABP. Transfected TM4 cells maintain the secretion of rat ABP for more than 40 days, with immunopositive rat ABP localized within cytoplasmic granules in the Golgi region and along cytoplasmic processes in TM4 transfected with either vector. Electron microscopic study revealed a higher development of cytoplasmic organelles involved in protein secretion.

  19. In vitro auxin binding to cellular membranes of cucumber fruits.

    PubMed

    Narayanan, K R; Mudge, K W; Poovaiah, B W

    1981-04-01

    Specific binding of 1-naphthaleneacetic acid (NAA) to crude membrane preparations from cucumber (Cucumis sativus L.) was demonstrated. This in vitro binding had a pH optimum of 3.75 and an equilibrium dissociation constant of 10 to 20 micromolar with 1250 picomoles binding sites per gram fresh weight. The NAA-binding sites were pronase sensitive. The supernatant from the fruit partially inhibited the in vitro NAA binding to fruit membranes. NAA, 2-naphthoxyacetic acid, 3-indoleacetic acid, 2-4-dichlorophenoxyacetic acid, and 2,3,5-triiodobenzoic acid, which are reported to be very good inducers of parthenocarpy in cucumber, showed a high degree of specific binding to cucumber fruit membranes. In comparison, 2-naphthaleneacetic acid and indolepropionic acid, which are reported to be very weak auxins in corn coleoptile, pea stem, and strawberry fruit growth bioassays, did not bind efficiently to cucumber fruit membranes. In vitro binding studies with fruit membranes suggest that auxin stimulated fruit growth may be mediated by membrane-associated, auxin-binding protein(s).

  20. NF-κB Enhances Androgen Receptor Expression through 5'-UTR Binding in Gingival Cells.

    PubMed

    Chang, J H; Wang, L F; Lu, S L; Huang, C F; Lu, H K

    2015-10-01

    Dihydropyridine-induced gingival overgrowth (DIGO) is a side effect observed in patients treated for hypertension. The disease is aggravated by inflammation. Nifedipine (Nif), a dihydropyridine, causes gingival overgrowth by increasing the expression of the androgen receptor (AR). Furthermore, the proinflammatory cytokine interleukin 1β (IL-1β) induces collagen α1(I) expression through the AR in DIGO fibroblasts. These observations prompted us to investigate whether and how nuclear factor kappa B (NF-κB) affects AR expression in DIGO. Therefore, gingival fibroblasts obtained from the tissues of patients with DIGO and healthy subjects were stimulated with IL-1β, Nif, or both. mRNA and protein expression was detected with real-time polymerase chain reaction and Western blotting. High correlation coefficients were observed for the mRNA expression of the AR, connective tissue growth factor, and collagen α1(I) induced by both drugs. Western blot analysis showed that IL-1β and Nif increased and activated NF-κB more in DIGO cells than in healthy cells. An electrophoretic mobility shift assay demonstrated that the promoter and 5'-untranslated regions (5'-UTRs) of the AR gene contains 3 binding sites for the NF-κB p65 subunit. A chromatin immunoprecipitation assay revealed that the NF-κB p65 subunit was associated with AR 5'-UTRs in gingival fibroblasts. A site-directed mutagenesis study indicated that a mutation of NF-κB binding sites reduced Nif- and IL-1β-induced AR promoter activities. Collectively, these data indicate that NF-κB is an essential transcriptional regulator of AR gene expression and thus plays a crucial role in collagen overproduction in DIGO fibroblasts.

  1. Dihydrotestosterone differentially modulates the mitogen-activated protein kinase and the phosphoinositide 3-kinase/Akt pathways through the nuclear and novel membrane androgen receptor in C6 cells.

    PubMed

    Gatson, Joshua W; Kaur, Paramjit; Singh, Meharvan

    2006-04-01

    Androgens such as dihydrotestosterone (DHT) are known to exert their effects through the activation of intracellular receptors that regulate the transcription of target genes. Alternatively, nongenomic mechanisms, including the activation of such signaling pathways as the MAPK pathways, have been described. It is unclear, however, whether this latter mechanism of action is mediated by the classical androgen receptor (AR) or some alternative mechanism. In this study, using a glial cell model (C6 cells) that we found to express the AR, we identified that DHT increased the phosphorylation of both ERK and Akt, key effectors of the neuroprotection-associated MAPK and phosphoinositide 3-kinase signaling pathways, respectively, and ERK phosphorylation was blocked by the AR antagonist, flutamide. In contrast, the membrane-impermeable, BSA-conjugated androgen (DHT-BSA) caused a dose-dependent suppression of ERK and Akt phosphorylation, suggesting the existence of a novel membrane-associated AR that mediates this opposite effect on neuroprotective signaling. This is also supported by the observation of DHT-displaceable binding sites on the cell surface of live C6 cells. Collectively, these data support the existence of a novel membrane-associated AR in glial cells and argue for the existence of two, potentially competing, pathways in a given cell or tissue. This mutual antagonism was supported by the ability of DHT-BSA to attenuate DHT-induced ERK phosphorylation. Thus, depending on the predominance of one receptor mechanism over another, the outcome of androgen treatment may be very different and, as such, could help explain existing discrepancies as to whether androgens are protective or damage inducing.

  2. Rabies virus binding to cellular membranes measured by enzyme immunoassay.

    PubMed

    Lentz, T L; Chester, J; Benson, R J; Hawrot, E; Tignor, G H; Smith, A L

    1985-05-01

    The binding of rabies virus to cellular membranes was measured using an enzyme-linked immunosorbent assay (ELISA). Virus binding to membranes adsorbed to the wells of microtiter plates was detected with rabies virus antibody and alkaline phosphatase-linked second antibody. The greatest degree of binding was to myotube, neuroblastoma, and salivary gland membranes; intermediate levels occurred in striated muscle and nerve membranes; and low levels of binding were found in other membranes, including those of most parenchymal organs. Binding of rabies virus to myotube membranes was saturable, dependent on pH (with an optimum of pH 6.0), facilitated by the divalent cations Ca++, Mn++, and Mg++, and was temperature dependent. Binding was greatly reduced by inactivation of virus with beta-propiolactone or treatment of virus with trypsin. In embryonic chick myotubes, total acetylcholine receptor content and acetylcholinesterase activity undergo marked changes during development, first increasing and then decreasing at the time of hatching. Binding of rabies virus followed a similar pattern, indicating that the virus may interact with the acetylcholine receptor or other surface molecules undergoing similar developmental changes.

  3. 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.

  4. Rapid bursts of androgen-binding protein (Abp) gene duplication occurred independently in diverse mammals

    PubMed Central

    2008-01-01

    Background The draft mouse (Mus musculus) genome sequence revealed an unexpected proliferation of gene duplicates encoding a family of secretoglobin proteins including the androgen-binding protein (ABP) α, β and γ subunits. Further investigation of 14 α-like (Abpa) and 13 β- or γ-like (Abpbg) undisrupted gene sequences revealed a rich diversity of developmental stage-, sex- and tissue-specific expression. Despite these studies, our understanding of the evolution of this gene family remains incomplete. Questions arise from imperfections in the initial mouse genome assembly and a dearth of information about the gene family structure in other rodents and mammals. Results Here, we interrogate the latest 'finished' mouse (Mus musculus) genome sequence assembly to show that the Abp gene repertoire is, in fact, twice as large as reported previously, with 30 Abpa and 34 Abpbg genes and pseudogenes. All of these have arisen since the last common ancestor with rat (Rattus norvegicus). We then demonstrate, by sequencing homologs from species within the Mus genus, that this burst of gene duplication occurred very recently, within the past seven million years. Finally, we survey Abp orthologs in genomes from across the mammalian clade and show that bursts of Abp gene duplications are not specific to the murid rodents; they also occurred recently in the lagomorph (rabbit, Oryctolagus cuniculus) and ruminant (cattle, Bos taurus) lineages, although not in other mammalian taxa. Conclusion We conclude that Abp genes have undergone repeated bursts of gene duplication and adaptive sequence diversification driven by these genes' participation in chemosensation and/or sexual identification. PMID:18269759

  5. Modeling androgen receptor flexibility: a binding mode hypothesis of CYP17 inhibitors/antiandrogens for prostate cancer therapy.

    PubMed

    Gianti, Eleonora; Zauhar, Randy J

    2012-10-22

    Prostate Cancer (PCa), a leading cause of cancer death worldwide (www.cancer.gov), is a complex malignancy where a spectrum of targets leads to a diversity of PCa forms. A widely pursued therapeutic target is the Androgen Receptor (AR). As a Steroid Hormone Receptor, AR serves as activator of transcription upon binding to androgens and plays a central role in the development of PCa. AR is a structurally flexible protein, and conformational plasticity of residues in the binding-pocket is a key to its ability to accommodate ligands from various chemical classes. Besides direct modulation of AR activity by antagonists, inhibition of cytochrome CYP17 (17α-hydroxylase/17,20-lyase), essential in androgen biosynthesis, has widely been considered an effective strategy against PCa. Interestingly, Handratta et al. (2005) discovered new, potent inhibitors of CYP17 (C-17 steroid derivatives) with pure AR antagonistic properties. Although the antiandrogenic activity of their lead compound (VN/124-1) has been experimentally proven both in vitro and in vivo, no structural data are currently available to elucidate the molecular determinants responsible for these desirable dual inhibitory properties. We implemented a Structure-based Drug Design (SBDD) approach to generate a valuable hypothesis as to the binding modes of steroidal CYP17 inhibitors/antiandrogens against the AR. To deal with the plasticity of residues buried in the Ligand Binding Domain (LBD), we developed a flexible-receptor Docking protocol based on Induced-Fit (IFD) methodology (www.schrodinger.com/). Our results constitute an ideal starting point for the rational design of next-generation analogues of CYP17 inhibitors/antiandrogens as well as an attractive tool to suggest novel chemical classes of AR antagonists.

  6. A sliding selectivity scale for lipid binding to membrane proteins

    PubMed Central

    Landreh, Michael; Marty, Michael T.; Gault, Joseph; Robinson, Carol V.

    2017-01-01

    Biological membranes form barriers that are essential for cellular integrity and compartmentalisation. Proteins that reside in the membrane have co-evolved with their hydrophobic lipid environment which serves as a solvent for proteins with very diverse requirements. As a result, membrane protein-lipid interactions range from completely non-selective to highly discriminating. Mass spectrometry (MS), in combination with X-ray crystallography and molecular dynamics simulations, enables us to monitor how lipids interact with intact membrane protein complexes and assess their effects on structure and dynamics. Recent studies illustrate the ability to differentiate specific lipid binding, preferential interactions with lipid subsets, and nonselective annular contacts. In this review, we consider the biological implications of different lipid-binding scenarios and propose that binding occurs on a sliding selectivity scale, in line with the view of biological membranes as facilitators of dynamic protein and lipid organization. PMID:27155089

  7. Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag

    PubMed Central

    Wen, Yi; Dick, Robert A.

    2016-01-01

    ABSTRACT The retroviral structural protein Gag binds to the inner leaflet of the plasma membrane (PM), and many cellular proteins do so as well. We used Rous sarcoma virus (RSV) Gag together with membrane sensors to study the principles governing peripheral protein membrane binding, including electrostatics, specific recognition of phospholipid headgroups, sensitivity to phospholipid acyl chain compositions, preference for membrane order, and protein multimerization. We used an in vitro liposome-pelleting assay to test protein membrane binding properties of Gag, the well-characterized MARCKS peptide, a series of fluorescent electrostatic sensor proteins (mNG-KRn), and the specific phosphatidylserine (PS) binding protein Evectin2. RSV Gag and mNG-KRn bound well to membranes with saturated and unsaturated acyl chains, whereas the MARCKS peptide and Evectin2 preferentially bound to membranes with unsaturated acyl chains. To further discriminate whether the primary driving force for Gag membrane binding is electrostatic interactions or preference for membrane order, we measured protein binding to giant unilamellar vesicles (GUVs) containing the same PS concentration in both disordered (Ld) and ordered (Lo) phases. RSV Gag and mNG-KRn membrane association followed membrane charge, independent of membrane order. Consistent with pelleting data, the MARCKS peptide showed preference for the Ld domain. Surprisingly, the PS sensor Evectin2 bound to the PS-rich Ld domain with 10-fold greater affinity than to the PS-rich Lo domain. In summary, we found that RSV Gag shows no preference for membrane order, while proteins with reported membrane-penetrating domains show preference for disordered membranes. IMPORTANCE Retroviral particles assemble on the PM and bud from infected cells. Our understanding of how Gag interacts with the PM and how different membrane properties contribute to overall Gag assembly is incomplete. This study examined how membrane charge and membrane order

  8. Demonstration of Auxin Binding to Strawberry Fruit Membranes 12

    PubMed Central

    Narayanan, Komaratchi R.; Mudge, Kenneth W.; Poovaiah, B. W.

    1981-01-01

    Presence of specific auxin-binding sites in strawberry fruit (Fragaria ananassa Duch. cv. Ozark Beauty) membranes has been demonstrated. These 1-naphthaleneacetic acid (NAA)-binding sites in the 80,000g to 120,000g fraction of the strawberry fruit membrane were pronase sensitive with an estimated equilibrium dissociation constant for NAA of 1.1 × 10−6 molar. The minimum concentration of NAA required to stimulate strawberry fruit growth was at least one order of magnitude higher than the minimum concentration of NAA required to stimulate corn coleoptile elongation. This was consistent with the higher equilibrium dissociation constant (lower affinity) for auxin binding to strawberry fruit membranes than to corn coleoptiles. Twelve auxin analogs, ranging from very strong to weak auxins, were tested for abilities to stimulate in situ strawberry fruit growth and to bind (displace or compete with NAA) to strawberry fruit membranes. The observed positive correlation (r = 0.74) between the in vitro binding to the 80,000 to 120,000 membrane fraction and the in situ biological activity of these analogs indicated that the NAA-binding sites in strawberry fruit membranes may represent physiologically relevant auxin receptors. PMID:16662094

  9. Polyester monomers lack ability to bind and activate both androgenic and estrogenic receptors as determined by in vitro and in silico methods.

    PubMed

    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.

  10. 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

    2013-08-02

    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.

  11. Dystrophin contains multiple independent membrane-binding domains.

    PubMed

    Zhao, Junling; Kodippili, Kasun; Yue, Yongping; Hakim, Chady H; Wasala, Lakmini; Pan, Xiufang; Zhang, Keqing; Yang, Nora N; Duan, Dongsheng; Lai, Yi

    2016-09-01

    Dystrophin is a large sub-sarcolemmal protein. Its absence leads to Duchenne muscular dystrophy (DMD). Binding to the sarcolemma is essential for dystrophin to protect muscle from contraction-induced injury. It has long been thought that membrane binding of dystrophin depends on its cysteine-rich (CR) domain. Here, we provide in vivo evidence suggesting that dystrophin contains three additional membrane-binding domains including spectrin-like repeats (R)1-3, R10-12 and C-terminus (CT). To systematically study dystrophin membrane binding, we split full-length dystrophin into ten fragments and examined subcellular localizations of each fragment by adeno-associated virus-mediated gene transfer. In skeletal muscle, R1-3, CR domain and CT were exclusively localized at the sarcolemma. R10-12 showed both cytosolic and sarcolemmal localization. Importantly, the CR-independent membrane binding was conserved in murine and canine muscles. A critical function of the CR-mediated membrane interaction is the assembly of the dystrophin-associated glycoprotein complex (DGC). While R1-3 and R10-12 did not restore the DGC, surprisingly, CT alone was sufficient to establish the DGC at the sarcolemma. Additional studies suggest that R1-3 and CT also bind to the sarcolemma in the heart, though relatively weak. Taken together, our study provides the first conclusive in vivo evidence that dystrophin contains multiple independent membrane-binding domains. These structurally and functionally distinctive membrane-binding domains provide a molecular framework for dystrophin to function as a shock absorber and signaling hub. Our results not only shed critical light on dystrophin biology and DMD pathogenesis, but also provide a foundation for rationally engineering minimized dystrophins for DMD gene therapy.

  12. Estrogens and Androgens Inhibit Association of RANKL with the Pre-osteoblast Membrane through Post-translational Mechanisms.

    PubMed

    Martin, Anthony; Yu, Jiali; Xiong, Jian; Khalid, Aysha B; Katzenellenbogen, Benita; Kim, Sung Hoon; Katzenellenbogen, John A; Malaivijitnond, Suchinda; Gabet, Yankel; Krum, Susan A; Frenkel, Baruch

    2017-02-18

    We have recently demonstrated that RUNX2 promoted, and 17-β-Estradiol (E2) diminished, association of RANKL with the cell membrane in pre-osteoblast cultures. Here we show that, similar to E2, dihydrotestosterone (DHT) diminishes association of RANKL and transiently transfected GFP-RANKL with the pre-osteoblast membrane without decreasing total RANKL mRNA or protein levels. Diminution of membrane-associated RANKL was accompanied with marked suppression of osteoclast differentiation from co-cultured pre-osteoclasts, even though DHT increased, not decreased, RANKL concentrations in pre-osteoblast conditioned media. A marked decrease in membrane-associated RANKL was observed after 30 minutes of either E2 or DHT treatment, and near-complete inhibition was observed by 1 hour, suggesting that the diminution of RANKL membrane association was mediated through non-genomic mechanisms. Further indicating dispensability of nuclear action of estrogen receptor, E2-mediated inhibition of RANKL membrane association was mimicked by an estrogen dendrimer conjugate (EDC) that cannot enter the cell nucleus. Finally, the inhibitory effect of E2 and DHT on RANKL membrane association was counteracted by the MMP inhibitor NNGH, and the effect of E2 (and not DHT) was antagonized by the Src inhibitor SU6656. Taken together, these results suggest that estrogens and androgens inhibit osteoblast-driven osteoclastogenesis through non-genomic, MMP-mediated RANKL dissociation from the cell membrane. This article is protected by copyright. All rights reserved.

  13. Inhibition of androgen metabolism and binding by a liposterolic extract of "Serenoa repens B" in human foreskin fibroblasts.

    PubMed

    Sultan, C; Terraza, A; Devillier, C; Carilla, E; Briley, M; Loire, C; Descomps, B

    1984-01-01

    We previously suggested [Steroids 33, (1979) 3; Steroids 37, (1981) 6] that cultured genital skin fibroblasts should prove useful for screening of potential antiandrogens in human and living target cells. "Serenoa repens" lipidic extract (S.R.E.) was recently reported (Br. J. Pharmacol., in press) to inhibit androgen action in animals. The present investigation was designed to study the antiandrogenicity of this compound in human cells: we therefore analyzed the effects of S.R.E. on the intracellular conversion of testosterone (T) to 5 alpha-reduced derivatives, and we investigated interaction of S.R.E. with the intracellular androgen-receptor complex. Since the chemical structure of the active component of S.R.E. is still unknown, results are expressed in U/ml (one unit is defined as the amount of S.R.E. required to inhibit 50% of the specific binding (IC50) of [3H]1881 to rat prostate cytosol). S.R.E. at different dilutions (5.7 to 28.6 U/ml) is added to culture media containing [3H]T or [3H]dihydrotestosterone (DHT) and incubated at 37 degrees C with cultured fibroblasts. 28.6 U/ml S.R.E. significantly alters the formation of DHT and strongly inhibits 3 ketosteroid reductase mediated conversion of DHT to 5 alpha-androstane-3 alpha, 17 beta-diol, characterized radiochemically by thin-layer chromatography. S.R.E. is a good competitor for the whole cell androgen receptor: 7.1 U/ml S.R.E. gives 50% inhibition of the binding of 2 X 10(-9) M [3H]DHT to its receptor. Competitive binding assays after cell fractionation indicate that S.R.E. is less potent in nuclear than in cytosol receptors. Sucrose gradient centrifugation of the radioactive cell lysate of fibroblasts demonstrates that 28.6 U/ml S.R.E. abolishes 70% of the 3.6 S receptor-complex radioactive peak. The present studies show that S.R.E. inhibits 5 alpha-reductase, 3-ketosteroid reductase and receptor binding of androgens in cultured human foreskin fibroblasts. As the search for the ideal antiandrogen

  14. Mechanisms of membrane deformation by lipid-binding domains.

    PubMed

    Itoh, Toshiki; Takenawa, Tadaomi

    2009-09-01

    Among an increasing number of lipid-binding domains, a group that not only binds to membrane lipids but also changes the shape of the membrane has been found. These domains are characterized by their strong ability to transform globular liposomes as well as flat plasma membranes into elongated membrane tubules both in vitro and in vivo. Biochemical studies on the structures of these proteins have revealed the importance of the amphipathic helix, which potentially intercalates into the lipid bilayer to induce and/or sense membrane curvature. Among such membrane-deforming domains, BAR and F-BAR/EFC domains form crescent-shaped dimers, suggesting a preference for a curved membrane, which is important for curvature sensing. Bioinformatics in combination with structural analyses has been identifying an increasing number of novel families of lipid-binding domains. This review attempts to summarize the evidence obtained by recent studies in order to gain general insights into the roles of membrane-deforming domains in a variety of biological events.

  15. Synthesis of 7alpha-(fluoromethyl)dihydrotestosterone and 7alpha-(fluoromethyl)nortestosterone, structurally paired androgens designed to probe the role of sex hormone binding globulin in imaging androgen receptors in prostate tumors by positron emission tomography.

    PubMed

    Parent, Ephraim E; Carlson, Kathryn E; Katzenellenbogen, John A

    2007-07-20

    Although prostate cancer growth is regulated by androgens through the androgen receptor (AR), in vitro assays of AR levels in prostate tumors have limited prognostic value. This might be improved by direct measurement of tumor AR in vivo using positron emission tomography (PET) imaging with fluorine-18-labeled androgens. Most AR PET imaging agents have been designed to limit steroid binding to serum proteins, but there is evidence that binding to sex hormone binding globulin (SHBG) might enhance tumor uptake. To probe the role of SHBG in prostate tumor uptake of PET imaging agents, we have synthesized two fluoro steroids, 7alpha-(fluoromethyl)dihydrotestosterone (7alpha-FM-DHT) and 7alpha-(fluoromethyl)nortestosterone (7alpha-FM-norT), by a route amenable to their labeling with [18F]fluoride ion. Both compounds have high affinity for AR, but 7alpha-FM-norT has much lower affinity for SHBG. Thus, these two fluoro steroids are well matched in terms of their site of fluorine labeling, similarity of structure, and equivalent AR binding affinity-but contrasting SHBG binding-and therefore can be used as agents for evaluating the role of SHBG binding in the target tissue uptake of AR PET imaging agents in humans.

  16. Sex hormone-binding globulin regulation of androgen bioactivity in vivo: validation of the free hormone hypothesis

    PubMed Central

    Laurent, Michaël R.; Hammond, Geoffrey L.; Blokland, Marco; Jardí, Ferran; Antonio, Leen; Dubois, Vanessa; Khalil, Rougin; Sterk, Saskia S.; Gielen, Evelien; Decallonne, Brigitte; Carmeliet, Geert; Kaufman, Jean-Marc; Fiers, Tom; Huhtaniemi, Ilpo T.; Vanderschueren, Dirk; Claessens, Frank

    2016-01-01

    Sex hormone-binding globulin (SHBG) is the high-affinity binding protein for androgens and estrogens. According to the free hormone hypothesis, SHBG modulates the bioactivity of sex steroids by limiting their diffusion into target tissues. Still, the in vivo physiological role of circulating SHBG remains unclear, especially since mice and rats lack circulating SHBG post-natally. To test the free hormone hypothesis in vivo, we examined total and free sex steroid concentrations and bioactivity on target organs in mice expressing a human SHBG transgene. SHBG increased total androgen and estrogen concentrations via hypothalamic-pituitary feedback regulation and prolonged ligand half-life. Despite markedly raised total sex steroid concentrations, free testosterone was unaffected while sex steroid bioactivity on male and female reproductive organs was attenuated. This occurred via a ligand-dependent, genotype-independent mechanism according to in vitro seminal vesicle organ cultures. These results provide compelling support for the determination of free or bioavailable sex steroid concentrations in medicine, and clarify important comparative differences between translational mouse models and human endocrinology. PMID:27748448

  17. Guanyl nucleotides modulate binding to steroid receptors in neuronal membranes.

    PubMed Central

    Orchinik, M; Murray, T F; Franklin, P H; Moore, F L

    1992-01-01

    The recently characterized corticosteroid receptor on amphibian neuronal membranes appears to mediate rapid, stress-induced changes in male reproductive behaviors. Because the transduction mechanisms associated with this receptor are unknown, we performed radioligand binding studies to determine whether this steroid receptor is negatively modulated by guanyl nucleotides. The binding of [3H]corticosterone to neuronal membranes was inhibited by nonhydrolyzable guanyl nucleotides in both equilibrium saturation binding and titration studies. The addition of guanyl nucleotide plus unlabeled corticosterone induced a rapid phase of [3H]corticosterone dissociation from membranes that was not induced by addition of unlabeled ligand alone. Furthermore, the equilibrium binding of [3H]corticosterone and the sensitivity of the receptor to modulation by guanyl nucleotides were both enhanced by Mg2+. These results are consistent with the formation of a ternary complex of steroid, receptor, and guanine nucleotide-binding protein that is subject to regulation by guanyl nucleotides. Therefore, rapid signal transduction through corticosteroid receptors on neuronal membranes appears to be mediated by guanine nucleotide-binding proteins. PMID:1570300

  18. Taurine allosterically modulates flunitrazepam binding to synaptic membranes.

    PubMed

    Quinn, M R; Miller, C L

    1992-09-01

    Taurine is hypothesized to exert its inhibitory neuromodulatory effects, in part, by interaction with the GABAA receptor. Although taurine displaces GABA agonist binding to synaptic membranes, its allosteric effects on the benzodiazepine recognition site of the GABAA receptor complex is unsettled. We determined the effects of taurine on [3H]flunitrazepam (Flu) binding to well-washed, frozen-thawed synaptic membranes prepared from rat cortex. Comparative binding studies were conducted at 37 degrees C and on ice (0-4 degrees C). At 37 degrees C taurine increased Flu binding in a concentration dependent way by interaction with a bicuculline sensitive site, similar to GABA. Taurine increased Flu binding by causing a decrease in KD. The maximal effectiveness of taurine on Flu binding could not be increased further by addition of GABA. In contrast, the maximal stimulation of Flu binding by GABA was decreased by addition of taurine to the level attained by taurine alone. These mixed agonist/antagonist effects of taurine are pharmacologically specific and qualify taurine as a partial GABA agonist in this type of allosteric interaction. However, taurine causes opposite effects on Flu binding when measured at 0-4 degrees C: taurine interacts with a bicuculline insensitive site to inhibit Flu binding by increasing the KD. Taurine inhibition of Flu binding is not overcome by increasing concentrations of GABA. Although the mechanism of taurine inhibition of Flu binding at 0-4 degrees C is unclear, it may be an indirect effect of taurine interaction with membrane phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Systematic and functional characterization of novel androgen receptor variants arising from alternative splicing in the ligand-binding domain

    PubMed Central

    Uo, T; Dvinge, H; Sprenger, C C; Bradley, R K; Nelson, P S; Plymate, S R

    2017-01-01

    The presence of intact ligand-binding domain (LBD) ensures the strict androgen-dependent regulation of androgen receptor (AR): binding of androgen induces structural reorganization of LBD resulting in release of AR from HSP90, suppression of nuclear export which otherwise dominates over import and nuclear translocation of AR as a transcription factor. Thus, loss or defects of the LBD abolish constraint from un-liganded LBD as exemplified by constitutively active AR variants (AR-Vs), which are associated with emerging resistance mechanism to anti-AR therapy in castration-resistant prostate cancer (mCRPC). Recent analysis of the AR splicing landscapes revealed mCRPC harboring multiple AR-Vs with diverse patterns of inclusion/exclusion of exons (exons 4–8) corresponding to LBD to produce namely exon-skipping variants. In silico construction for these AR-Vs revealed four novel AR-Vs having unique features: Exclusion of specified exons introduces a frameshift in variants v5es, v6es and v7es. ARv56es maintains the reading frame resulting in the inclusion of the C-terminal half of the LBD. We systematically characterized these AR-Vs regarding their subcellular localization, affinity for HSP90 and transactivation capability. Notably, ARv5es was free from HSP90, exclusively nuclear, and constitutively active similarly as previously reported for v567es. In contrast, v6es and v7es were similar in that they are cytoplasmic, transcriptionally inactive and bind HSP90, ARv56es was present in both nucleus and cytoplasm, does not bind HSP90 and is transcriptionally inactive. Converting these transcriptionally inactive AR-Vs into active forms, we identified the two separate elements that allosterically suppress otherwise constitutively active AR-Vs; one in exon 5 for v6es and v7es and the other in exon 8 for v56es. Our findings identify a novel constitutively active AR-V, ARv5es and establish a method to predict potential activities of AR-Vs carrying impaired LBD. PMID:27694897

  20. Direct Membrane Binding by Bacterial Actin MreB

    PubMed Central

    Salje, Jeanne; van den Ent, Fusinita; de Boer, Piet; Löwe, Jan

    2011-01-01

    Summary Bacterial actin MreB is one of the key components of the bacterial cytoskeleton. It assembles into short filaments that lie just underneath the membrane and organize the cell wall synthesis machinery. Here we show that MreB from both T. maritima and E. coli binds directly to cell membranes. This function is essential for cell shape determination in E. coli and is proposed to be a general property of many, if not all, MreBs. We demonstrate that membrane binding is mediated by a membrane insertion loop in TmMreB and by an N-terminal amphipathic helix in EcMreB and show that purified TmMreB assembles into double filaments on a membrane surface that can induce curvature. This, the first example of a membrane-binding actin filament, prompts a fundamental rethink of the structure and dynamics of MreB filaments within cells. PMID:21816350

  1. Repair of Nerve Cell Membrane Damage by Calcium-Dependent, Membrane-Binding Proteins (Revised)

    DTIC Science & Technology

    2012-09-01

    family exhibit a “ bivalent ” activity resulting in the aggregation of membranes coincident with the binding of the annexin to the membrane [7,8]. Such...10. [12] D.J. Selkoe, Alzheimer’s disease: genes , proteins, and therapy, Physiol Rev 81 (2001) 741-766. [13] A. Demuro, I. Parker, G.E. Stutzmann

  2. Binding constants of membrane-anchored receptors and ligands depend strongly on the nanoscale roughness of membranes.

    PubMed

    Hu, Jinglei; Lipowsky, Reinhard; Weikl, Thomas R

    2013-09-17

    Cell adhesion and the adhesion of vesicles to the membranes of cells or organelles are pivotal for immune responses, tissue formation, and cell signaling. The adhesion processes depend sensitively on the binding constant of the membrane-anchored receptor and ligand proteins that mediate adhesion, but this constant is difficult to measure in experiments. We have investigated the binding of membrane-anchored receptor and ligand proteins with molecular dynamics simulations. We find that the binding constant of the anchored proteins strongly decreases with the membrane roughness caused by thermally excited membrane shape fluctuations on nanoscales. We present a theory that explains the roughness dependence of the binding constant for the anchored proteins from membrane confinement and that relates this constant to the binding constant of soluble proteins without membrane anchors. Because the binding constant of soluble proteins is readily accessible in experiments, our results provide a useful route to compute the binding constant of membrane-anchored receptor and ligand proteins.

  3. Disorders of androgen action.

    PubMed

    Sultan, Charles; Lumbroso, Serge; Paris, Françoise; Jeandel, Claire; Terouanne, B; Belon, Charles; Audran, F; Poujol, N; Georget, V; Gobinet, J; Jalaguier, S; Auzou, G; Nicolas, J C

    2002-08-01

    Disorders of androgen action are the main cause of male pseudohermaphroditism and include 5alphaR deficiency and androgen receptor defects. 5alphaR deficiency is characterized by female genitalia with some degree of masculinization, clitoromegaly, and severely bifid scrotum corresponding to the so-called pseudovaginal perineoscrotal hypospadias. At the onset of puberty, increased muscle mass, development of pubic hair, and phallic growth are associated with the acquisition of male gender identity. Normal or increased levels of testosterone and an elevated testosterone-to-dihydrotestosterone ratio after human chorionic gonadotropin stimulation testing suggest 5alphareductase deficiency, and the diagnosis can be ascertained by identifying the mutation in the 5alphaR-2 gene. Whatever the patient's age at diagnosis, psychological evaluation with 5alphaRD is vital. Androgen receptor defects encompass two clinical expressions: the complete and partial androgen insensitivity syndromes. Complete androgen insensitivity syndrome should be suspected at birth in the presence of inguinal hernia in a girl without genital ambiguity. At puberty, the sign of alert is primary amenorrhea with normal female phenotype and harmonious mammary development but no pubic hair growth. Partial androgen insensitivity syndrome covers a wide spectrum of undervirilized phenotypes ranging from clitoromegaly at birth to infertile men. In all cases, complementary investigations should include plasma testosterone and luteinizing hormone as well as androgen-binding capacity in cultured genital skin fibroblasts. Diagnosis is confirmed by identification of the androgen receptor gene mutation. Although patients with complete androgen insensitivity syndrome are raised as females, patients with partial androgen insensitivity syndrome should be managed according to age at diagnosis, response to treatment with exogenous androgens, and the presence of an androgen gene mutation. Gonadectomy in complete androgen

  4. 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

  5. (/sup 3/H) clonidine binding to rat hippocampal membranes

    SciTech Connect

    George, C.R.

    1982-02-01

    Alpha adrenergic receptor subtypes in rat hippocampal membranes were studied, using (/sup 3/H) clonidine as the radioactive ligand. On the basis of competitive binding studies, using the selective antagonist-prazosin, WB-4101, and yohimbine, (/sup 3/H) clonidine appeared to bind to a population of presynaptic sites that are pharmacologically similar to receptors previously classified as alpha 2. A computerized model that linearized and produced the best possible fit to the experimental data points indicated that (/sup 3/H) clonidine binds to a single population of receptors possessing equal affinity for the ligand. Binding data also indicated that rat hippo-campus contains significantly fewer (/sup 3/H)clonidine binding sites than rat cortex.

  6. Binding assays with artificial tethered membranes using surface plasmon resonance.

    PubMed

    Wiltschi, Birgit; Knoll, Wolfgang; Sinner, Eva-Kathrin

    2006-06-01

    Surface sensitive optical techniques based on surface plasmon resonance have become interesting for biosciences in the context of biorecognition and binding studies at functional surfaces. We use surface plasmon resonance spectroscopy (SPS) in combination with surface plasmon enhanced fluorescence spectroscopy (SPFS) for the characterization of interaction processes associated with biomembranes. The biological membrane is mimicked by a tethered membrane consisting of a planar lipid bilayer attached to a gold surface via a hydrophilic anchor peptide. The interaction between membrane-bound hydrophobic compounds and free hydrophilic molecules is monitored in real-time and with high sensitivity and selectivity by combined SPS/SPFS. In this review we shortly discuss the principles of surface plasmon resonance and its utilization in SPS and SPFS. A detailed description of the required instrumentation for combined SPS and SPFS is presented. Furthermore, we outline the design of a binding assay with a tethered bilayer and the procedure of the artificial membrane system built-up is delineated. We also present examples that demonstrate the potential of combined SPS/SPFS assays with artificial tethered membranes. The method provides insight into the interaction of integral membrane proteins with various hydrophilic ligands and the specific recognition of small lipophilic molecules by soluble proteins.

  7. 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 ...

  8. Polycation binding to glomerular basement membrane. Effect of biochemical modification.

    PubMed

    Bertolatus, J A; Hunsicker, L G

    1987-02-01

    The polycation hexadimethrine (HDM) binds to anionic sites in the glomerular basement membrane (GBM) and causes heavy proteinuria when infused in vivo. An in vitro assay of 3H-HDM binding to isolated dog GBM was developed, to permit further analysis of the GBM components binding HDM. 3H-HDM binding to isolated GBM was saturable, reversible in dose-dependent fashion by competing polycations, and inhibited by increasing salt concentration and low pH. The pH dependence of binding suggested that most of the HDM binds to carboxyl groups rather than to the sulfate groups of proteoglycans. Removal of heparan sulfate by heparinase or purified heparatinase had no detectable effect on HDM binding. Treatment of GBM with neuraminidase, hyaluronidase, or chondroitinase reduced binding of HDM by a maximum of 20 to 38%. However, substitution of carboxyl anions with nonionizable glycine methyl ester residues resulted in complete elimination of HDM binding. Parallel results were obtained in studies of glomerular localization of cationized ferritin (CatF), pI 8.5. After carboxyl substitution, GBM did not bind CatF; heparinase-treated GBM bound CatF in a distribution not demonstrably different from normal. Cellulose acetate electrophoresis of glycosaminoglycan fractions prepared from treated GBM confirmed that carboxyl modification did not alter the content or charge of the heparan sulfate of GBM, but heparinase treatment removed at least 90% of heparan sulfate. The results indicate that carboxyl groups are quantitatively more important than heparan sulfate for binding of HDM in vitro. Since HDM causes proteinuria in vivo, carboxyl groups may be important for maintenance of normal permselectivity.

  9. Androgen responsiveness of the murine beta-glucuronidase gene is associated with nuclease hypersensitivity, protein binding, and haplotype-specific sequence diversity within intron 9.

    PubMed Central

    Lund, S D; Gallagher, P M; Wang, B; Porter, S C; Ganschow, R E

    1991-01-01

    The tissue specificity and genetic variability of the murine beta-glucuronidase (GUS) response to androgen provide useful markers for identifying elements which underlie this responsiveness. While GUS is expressed constitutively in all examined cell types, kidney epithelial cells uniquely exhibit a manyfold yet slow rise in GUS mRNA and enzyme levels when stimulated by androgens. Three major phenotypes of this androgen response have been described among inbred strains of mice: (i) a strong response in strains of the Gusa haplotype, (ii) a reduced response in strains of the Gusb and Gush haplotypes, and (iii) no response, as observed in Gusor mice. These response variants define a cis-active element(s) which is tightly linked to the GUS structural gene. Nuclease hypersensitivity scans of kidney chromatin within and surrounding the structural gene revealed an androgen-inducible hypersensitive site in intron 9 of the gene in Gusa but not in Gusor mice. When a radiolabeled fragment of Gusa DNA containing this hypersensitive site was incubated with kidney nuclear extracts and then subjected to gel electrophoresis, two shifted bands were observed whose levels were dramatically higher in extracts of androgen-treated than in those of untreated Gusa mice. The shifted bands reflect binding of a kidney-specific factor(s) to a 57-bp region of complex dyad symmetry in Gusa and Gusor mice which is partially deleted in Gusb and Gush mice. This binding site is located approximately 130 bp downstream of a glucocorticoid response element sequence motif which is totally deleted in [Gus]or mice. Taken together, our results suggest that the androgen responsiveness of GUS in murine kidney epithelial cells is controlled by elements within the proximal end of intron 9 of the GUS structural gene. Images PMID:1922055

  10. Binding of bovine factor Va to phosphatidylcholine membranes.

    PubMed Central

    Koppaka, V; Lentz, B R

    1996-01-01

    The interaction of bovine factor Va with phosphatidylcholine membranes was examined using four different fluorescence techniques: 1) changes in the fluorescence anisotropy of the fluorescent membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to monitor the interaction of factor Va with 1,2-dimyristoyl-3-sn-phosphatidylcholine (DMPC) small unilamellar vesicles (SUVs), 2) changes in the fluorescence anisotropy of N-(lissamine rhodamine B sulfonyl) diacyl phosphati-dylethanolamine (Rh-PE) incorporated into SUVs prepared from 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC), 3) changes in the fluorescence anisotropy of fluorescein-labeled factor Va (labeled in the heavy chain) upon interaction with POPC SUVs, 4) fluorescence energy transfer from fluorescein-labeled factor Va to rhodamine-labeled POPC SUVs. In the first two sets of experiments, labeled lipid vesicles were titrated with unlabeled protein, whereas, in the latter two types of experiments, labeled factor Va was titrated with vesicles. For the weak binding observed here, it was impossible from any one binding experiment to obtain precise estimates of the three parameters involved in modeling the lipid-protein interaction, namely, the dissociation constant Kd, the stoichiometry of binding i, and the saturation value of the observable Rmax from any one experiment. However, a global analysis of the four data sets involving POPC SUVs yielded a stable estimate of the binding parameters (Kd of approximately 3.0 microM and a stoichiometry of approximately 200 lipids per bound factor Va). Binding to DMPC SUVs may be of slightly higher affinity. These observations support the contention that association of factor Va with a membrane involves a significant acidic-lipid-independent interaction along with the more commonly accepted acidic-lipid-dependent component of the total binding free energy. PMID:8744331

  11. 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.

  12. Tight binding of proteins to membranes from older human cells.

    PubMed

    Truscott, Roger J W; Comte-Walters, Susana; Ablonczy, Zsolt; Schwacke, John H; Berry, Yoke; Korlimbinis, Anastasia; Friedrich, Michael G; Schey, Kevin L

    2011-12-01

    The lens is an ideal model system for the study of macromolecular aging and its consequences for cellular function, since there is no turnover of lens fibre cells. To examine biochemical processes that take place in the lens and that may also occur in other long-lived cells, membranes were isolated from defined regions of human lenses that are synthesised at different times during life, and assayed for the presence of tightly bound cytosolic proteins using quantitative iTRAQ proteomics technology. A majority of lens beta crystallins and all gamma crystallins became increasingly membrane bound with age, however, the chaperone proteins alpha A and alpha B crystallin, as well as the thermally-stable protein, βB2 crystallin, did not. Other proteins such as brain-associated signal protein 1 and paralemmin 1 became less tightly bound in the older regions of the lens. It is evident that protein-membrane interactions change significantly with age. Selected proteins that were formerly cytosolic become increasingly tightly bound to cell membranes with age and are not removed even by treatment with 7 M urea. It is likely that such processes reflect polypeptide denaturation over time and the untoward binding of proteins to membranes may alter membrane properties and contribute to impairment of communication between older cells.

  13. 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.

  14. The size and detergent binding of membrane proteins.

    PubMed

    Clarke, S

    1975-07-25

    Sucrose density gradient centrifugation has been used to measure the binding of Triton X-100 above its critical micellar concentration to a variety of purified membrane and non-membrane proteins. In addition, binding studies were done on the three proteins below the critical micellar concentration of detergent to distinguish between the interaction of proteins with detergent monomers and detergent micelles. A procedure is described for the calculation of the molecular weight of these Triton X-100 protein complexes and measurements were made for opsin, plasma low density lipoprotein, the (Na-+ plus K-+)-dependent adenosine triphosphatase, the human red blood cell major sialoglycoprotein (PAS-1) and the human red blood cell minor glycoprotein (bandIII). These proteins behave as monomers or dimers in detergent and bind between 0.28 and 1.12 g of detergent per g of protein. A general method is also present for calculating the molecular size and shape of impure membrane proteins in detergent. Finally, Triton X-100 was shown to replace bound Na dodecyl-SO4 on the minor glycoprotein of the red blood cell.

  15. Androgens act synergistically to enhance estrogen-induced upregulation of human tissue kallikreins 10, 11, and 14 in breast cancer cells via a membrane bound androgen receptor.

    PubMed

    Paliouras, Miltiadis; Diamandis, Eleftherios P

    2008-04-01

    The regulation of gene expression by steroid hormones plays an important role in the normal development and function of many organs, as well as in the pathogenesis of endocrine-related cancers, especially breast cancer. However, clinical data suggest that combined testosterone and estrogen treatments on post-menopausal women increase the risk of breast cancer. Experiments have shown that many, if not all kallikreins are under steroid hormone regulation in breast cancer cell lines. Their implication as prognostic and diagnostic markers has also been well-documented. Thus, we investigated the effect of combined hormone stimulation with androgens and 17beta-estradiol on the ductal caricinoma cell line BT474. This cell line has been shown to be sensitive to both, androgens (secreting PSA) and estrogens (secreting a number of kallikreins including KLK10, 11, and KLK14). We found that PSA expression was downregulated upon combined hormone stimulation, confirming reports that estrogen can antagonize and block the activity of the androgen receptor. Upon analysis of estrogen-sensitive kallikreins 10, 11, and 14, all showed to be synergistically enhanced in their expression three- to fourfold, upon joint hormone treatment versus individual hormone stimulation. The enhancement is dependent upon the action of androgens as treatment with the androgen receptor antagonist cyproterone actetate normalized the expression of KLK10, 11, and KLK14 to estrogen-stimulation levels. The synergistic effects between estrogens and androgens on estrogen-sensitive genes may have implications on the role of the kallikreins in associated risk of breast cancer and progression.

  16. Discovery of small-molecule inhibitors selectively targeting the DNA-binding domain of the human androgen receptor.

    PubMed

    Li, Huifang; Ban, Fuqiang; Dalal, Kush; Leblanc, Eric; Frewin, Kate; Ma, Dennis; Adomat, Hans; Rennie, Paul S; Cherkasov, Artem

    2014-08-14

    The human androgen receptor (AR) is considered as a master regulator in the development and progression of prostate cancer (PCa). As resistance to clinically used anti-AR drugs remains a major challenge for the treatment of advanced PCa, there is a pressing need for new anti-AR therapeutic avenues. In this study, we identified a binding site on the DNA binding domain (DBD) of the receptor and utilized virtual screening to discover a set of micromolar hits for the target. Through further exploration of the most potent hit (1), a structural analogue (6) was identified demonstrating 10-fold improved anti-AR potency. Further optimization resulted in a more potent synthetic analogue (25) with anti-AR potency comparable to a newly FDA-approved drug Enzalutamide. Site-directed mutagenesis demonstrated that the developed inhibitors do interact with the intended target site. Importantly, the AR DBD inhibitors could effectively inhibit the growth of Enzalutamide-resistant cells as well as block the transcriptional activity of constitutively active AR splice variants, such as V7.

  17. Selection shaped the evolution of mouse androgen-binding protein (ABP) function and promoted the duplication of Abp genes.

    PubMed

    Karn, Robert C; Laukaitis, Christina M

    2014-08-01

    In the present article, we summarize two aspects of our work on mouse ABP (androgen-binding protein): (i) the sexual selection function producing incipient reinforcement on the European house mouse hybrid zone, and (ii) the mechanism behind the dramatic expansion of the Abp gene region in the mouse genome. Selection unifies these two components, although the ways in which selection has acted differ. At the functional level, strong positive selection has acted on key sites on the surface of one face of the ABP dimer, possibly to influence binding to a receptor. A different kind of selection has apparently driven the recent and rapid expansion of the gene region, probably by increasing the amount of Abp transcript, in one or both of two ways. We have shown previously that groups of Abp genes behave as LCRs (low-copy repeats), duplicating as relatively large blocks of genes by NAHR (non-allelic homologous recombination). The second type of selection involves the close link between the accumulation of L1 elements and the expansion of the Abp gene family by NAHR. It is probably predicated on an initial selection for increased transcription of existing Abp genes and/or an increase in Abp gene number providing more transcriptional sites. Either or both could increase initial transcript production, a quantitative change similar to increasing the volume of a radio transmission. In closing, we also provide a note on Abp gene nomenclature.

  18. 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

  19. Photoaffinity labeling of uncoupler binding sites on mitochondrial membrane.

    PubMed

    Kurup, C K; Sanadi, D R

    1977-02-01

    3H 2-azido-4-nitrophenol, a photoactive uncoupler, has been synthesized, and its uncoupling action on oxidative phosphorylation and its binding to the mitochondrial membrane have been studied. The uncoupler bound covalently to the mitochondrial membrane on photoirradiation was 3-4 times that bound reversibly in the absence of light. When irradiation was carried out in the presence of serum albumin, covalent binding was significantly depressed. The pattern of loss of ATP-Pi exchange activity with increasing amounts of the uncoupler suggests that serum albumin prevents the binding of the uncoupler to the functional sites as well. Polyacrylamide gel electrophoresis of photoaffinity labeled submitochondrial particles in the presence of sodium dodecyl sulfate revealed that a 9000 dalton peptide bound high levels of uncoupler. Other proteins in the molecular weight range of 20,000-40,000 and 55,000 were also labeled. Photolysis in the presence of serum albumin or ATP decreased the covalent binding of the uncoupler to all the proteins, but particularly to the 20,000 dalton component. Soluble ATPase and the mitochondrial proteolipid purified from labeled mitochondria showed the presence of label.

  20. Androgens Regulate T47D Cells Motility and Invasion through Actin Cytoskeleton Remodeling

    PubMed Central

    Montt-Guevara, Maria Magdalena; Shortrede, Jorge Eduardo; Giretti, Maria Silvia; Giannini, Andrea; Mannella, Paolo; Russo, Eleonora; Genazzani, Alessandro David; Simoncini, Tommaso

    2016-01-01

    The relationship between androgens and breast cancer is controversial. Androgens have complex effects on breast cancer progression and metastasis. Moreover, androgen receptor (AR) is expressed in approximately 70 to 90% of invasive breast carcinomas, which has prognostic relevance in basal-like cancers and in triple-negative breast cancers. Recent studies have associated the actin-binding proteins of the ezrin–radixin–moesin (ERM) family with metastasis in endocrine-sensitive cancers. We studied on T47D breast cancer cells whether androgens with different characteristics, such as testosterone (T), dihydrotestosterone (DHT), and dehydroepiandrosterone (DHEA) may regulate breast cancer cell motility and invasion through the control of actin remodeling. We demonstrate that androgens promote migration and invasion in T47D via Moesin activation. We show that T and DHEA exert their actions via the AR and estrogen receptor (ER), while the non-aromatizable androgen – DHT – only recruits AR. We further report that androgen induced significant changes in actin organization with pseudopodia along with membrane ruffles formation, and this process is mediated by Moesin. Our work identifies novel mechanisms of action of androgens on breast cancer cells. Through the modulation of Moesin, androgens alter the architecture of cytoskeleton in T47D breast cancer cell and promote cell migration and invasion. These results could help to understand the biological actions of androgens on breast cancer and, eventually, to develop new strategies for breast cancer treatment. PMID:27746764

  1. Binding of white spot syndrome virus to Artemia sp. cell membranes.

    PubMed

    Feng, Shuying; Li, Guangda; Feng, Wenpo; Huang, Jie

    2013-10-01

    Using differential velocity centrifugation, cell membranes of Artemia sp. were prepared, and their binding to white spot syndrome virus (WSSV) was analyzed in vitro. The results indicated that WSSV can specifically bind to Artemia cell membranes, and that WSSV receptor very likely existed in this membrane, which suggested that Artemia sp. may be a reservoir of WSSV. This study investigated the specific WSSV binding site by performing competitive inhibition experiments using shrimp gill cell membranes to bind WSSV to Artemia cell membranes. The results showed that shrimp gill cell membranes had a distinct inhibition effect on the specific binding of Artemia cell membranes to WSSV. Thus, potentially similar WSSV receptors or binding sites existed on Artemia sp. cell membranes and shrimp gill cell membranes. Taken together, these findings may provide experimental basis for the development of an effective approach to controlling WSSV, and theoretical basis for the study of WSSV receptors.

  2. 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

  3. 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

  4. 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.

  5. Rat androgen-binding protein: evidence for identical subunits and amino acid sequence homology with human sex hormone-binding globulin.

    PubMed

    Joseph, D R; Hall, S H; French, F S

    1987-01-01

    The cDNA for rat androgen-binding protein (ABP) was previously isolated from a bacteriophage lambda gt11 rat testis cDNA library and its identity was confirmed by epitope selection. Hybrid-arrested translation studies have now demonstrated the identity of the isolates. The nucleotide sequence of a near full-length cDNA encodes a 403-amino acid precursor (Mr = 44,539), which agrees in size with the cell-free translation product (Mr = 45,000) of ABP mRNA. Putative sites of N-glycosylation and signal peptide cleavage were identified. Comparison of the predicted amino acid sequence of rat ABP with the amino-terminal amino acid sequence of human sex hormone-binding globulin revealed that 17 of 25 residues are identical. On the basis of the predicted amino acid sequence the molecular weight of the primary translation product, lacking the signal peptide, was 41,183. Hybridization analyses indicated that the two subunits of ABP are coded for by a single gene and a single mRNA species. Our results suggest that ABP consists of two subunits with identical primary sequences and that differences in post-translational processing result in the production of 47,000 and 41,000 molecular weight monomers.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. The effect of vesicle shape, line tension, and lateral tension on membrane-binding proteins

    NASA Astrophysics Data System (ADS)

    Hutchison, Jaime B.

    Model membranes allow for the exploration of complex biological phenomena with simple, controllable components. In this thesis we employ model membranes to determine the effect of vesicle properties such as line tension, lateral tension, and shape on membrane-binding proteins. We find that line tension at the boundary between domains in a phase separated vesicle can accumulate model membrane-binding proteins (green fluorescent protein with a histidine tag), and that those proteins can, in turn, alter vesicle shape. These results suggest that domains in biological membranes may enhance the local concentration of membrane-bound proteins and thus alter protein function. We also explore how membrane mechanical and chemical properties alter the function of the N-BAR domain of amphiphysin, a membrane-binding protein implicated in endocytosis. We find that negatively charged lipids are necessary for N-BAR binding to membranes at detectable levels, and that, at least for some lipid species, binding may be cooperative. Measurements of N-BAR binding as a function of vesicle tension reveal that modest membrane tension of around 2 mN/m, corresponding to a strain of around 1%, strongly increases N-BAR binding. We attribute this increase in binding with tension to the insertion of N-BAR's N-terminal amphipathic helix into the membrane which increases the membrane area. We propose that N-BAR, which was previously described as being able to sense membrane curvature, may be sensing strain instead. Measurements of membrane deformation by N-BAR as a function of membrane tension reveal that tension can hinder membrane deformation. Thus, tension may favor N-BAR binding yet suppress membrane deformation/tubulation, which requires work against tension. These results suggest that membrane tension, a parameter that is often not controlled in model membranes but is tightly controlled in biological cells, may be important in regulating protein binding and assembly and, hence, protein

  11. 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

  12. Synaptotagmin-1 binds to PIP(2)-containing membrane but not to SNAREs at physiological ionic strength.

    PubMed

    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

    2015-10-01

    The Ca(2+) 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's binding to membranes or SNAREs has not been explored. Here we show that binding of rat synaptotagmin-1 to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2) is regulated by charge shielding caused by the presence of divalent cations. Surprisingly, polyvalent ions such as ATP and Mg(2+) completely abrogate synaptotagmin-1 binding to SNAREs regardless of the presence of Ca(2+). Altogether, our data indicate that at physiological ion concentrations Ca(2+)-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.

  13. The mouse salivary androgen-binding protein (ABP) gene cluster on chromosomes 7: characterization and evolutionary relationships.

    PubMed

    Laukaitis, Christina M; Dlouhy, Stephen R; Karn, Robert C

    2003-10-01

    Mouse salivary androgen-binding protein (ABP) is a pair of dimers, composed of an alpha subunit disulfide bridged to either a beta or a gamma subunit. It has been proposed that each subunit is encoded by a distinct gene: Abpa, Abpb, and Abpg for the alpha, beta, and gamma subunits, respectively. We report here the structures and sequences of the genes that encode these three subunits. Each gene has three exons separated by two introns. Mouse salivary ABP is a member of the secretoglobin family, and we compare the structure of the three ABP subunit genes to those of 18 other mammalian secretoglobins. We map the three genes as a gene cluster located 10 cM from the centromere of Chromosome (Chr) 7 and show that Abpa is the closest of the three to the gene for glucose phosphate isomerase (GPI) and that Abpg is the closest to the centromere, with Abpb mapping between them. Abpa is oriented in the opposite direction to Abpb and Abpg, with its 5' end directed toward their 5' ends. We compare the location of these genes with other secretoglobin genes in the mouse genome and with the known locations of secretoglobin genes in the human genome and present evidence that strong positive selection has driven the divergence of the coding regions of Abpb and Abpg since the putative duplication event that created them.

  14. Membrane solubilization by a hydrophobic polyelectrolyte: surface activity and membrane binding.

    PubMed Central

    Thomas, J L; Barton, S W; Tirrell, D A

    1994-01-01

    We have previously observed that the hydrophobic polyelectrolyte poly(2-ethylacrylic acid) solubilizes lipid membranes in a pH-dependent manner, and we have exploited this phenomenon to prepare lipid vesicles that release their contents in response to pH, light, or glucose (Thomas, J. L., and D. A. Tirrell. Acc. Chem. Res. 25:336-342, 1992). The physical basis for the interaction between poly(2-ethylacrylic acid) and lipid membranes has been explored using surface tensiometry and fluorimetry. Varying the polymer concentration results in changes in surface activity and membrane binding that correlate with shifts in the critical pH for membrane solubilization. Furthermore, the binding affinity is reduced as the amount of bound polymer increases. These results are consistent with a hydrophobically driven micellization process, similar to those observed with apolipoproteins, melittin, and other amphiphilic alpha-helix-based polypeptides. The absence of specific secondary structure in the synthetic polymer suggests that amphiphilicity, rather than structure, is the most important factor in membrane micellization by macromolecules. PMID:7811920

  15. Protein binding properties of surface-modified porous polyethylene membranes.

    PubMed

    Greene, George; Radhakrishna, Harish; Tannenbaum, Rina

    2005-10-01

    In this study, we quantified the adsorption of immunoglobulin G (IgG) protein onto several polyelectrolyte-modified sintered porous polyethylene (PPE) membranes. The polymer surfaces had both cationic and anionic charges obtained via the adsorption of polyethylenimine (PEI) and polyacrylic acid (PAA), respectively, onto plasma-activated PPE. The amount of IgG adsorption was determined by measuring the gamma radiation emitted by [125I]-IgG radio labeled protein. By studying the impact of pH and ionic strength on IgG adsorption, we attempted to characterize the role and nature of the electrostatic interactions involved in the adsorption process to better understand how these interactions were influenced by the charge and structure of immobilized polyelectrolyte complexes at modified membrane surfaces. We were able to show that surface modification of PPE membranes with adsorbed PEI monolayers and PEI-PAA bilayers can greatly improve the IgG binding ability of the membrane under optimized conditions. We also showed that the observed improvement in the IgG binding is derived from electrostatic interactions between IgG and the polyelectrolyte surface. In addition, we found that the greatest IgG adsorption occurred when the IgG and the surface possessed predominantly opposite charges, rather than when the surface possessed the greatest electrostatic charge. Finally, we have found that the molecular weight of the terminating polyelectrolyte has a noticeable effect upon the electrostatic interactions between IgG and the PEI-PAA bilayer-modified PPE surfaces.

  16. Specific binding of GM1-binding peptides to high-density GM1 in lipid membranes.

    PubMed

    Matsubara, Teruhiko; Iijima, Kazutoshi; Nakamura, Miwa; Taki, Takao; Okahata, Yoshio; Sato, Toshinori

    2007-01-16

    The ganglioside Galbeta1-3GalNAcbeta1-4(Neu5Acalpha2-3)Galbeta1-4Glcbeta1-1'Cer (GM1) is an important receptor. We have previously identified GM1-binding peptides based on affinity selection from a random peptide library. In the present study, we determined the amino acids essential for binding GM1 and investigated the specific interaction with GM1 in the lipid membrane. Arginines and aromatic amino acids in the consensus sequence (W/F)RxL(xP/Px)xFxx(Rx/xR)xP contributed to the ability of the peptides to bind GM1. The peptide p3, VWRLLAPPFSNRLLP, having the consensus sequence, showed high affinity for GM1 with a dissociation constant of 1.2 microM. Furthermore, the density-dependent binding of p3 was investigated using mixed monolayers of GM1 and Glcbeta1-1'Cer (GlcCer). p3 binds preferentially to high-density GM1, and its interaction with GM1 was found to be cooperative based on a Hill plot. These results indicated that a lateral assembly of GM1 molecules was required for the recognition of carbohydrates by p3. The GM1-binding peptide played a role as a unique anti-GM1 probe differing from the cholera toxin B subunit or antibodies.

  17. The Hinge Region as a Key Regulatory Element of Androgen Receptor Dimerization, DNA Binding, and Transactivation

    DTIC Science & Technology

    2005-05-01

    led to a structure depicted in figure 1A. Two zinc coordinating modules that constitute the receptors DNA-binding domain, are involved in the...expression plasmid (reviewed in Claessens et al. 2001). This led first to the description of the PB-ARE-2 (Claessens et al. 1996), later of scARE and...constructs for specific mutants: has been done and is ongoing. This has led to most of the observations reported in section II of this report. iii.c

  18. Androgen resistance.

    PubMed

    Hughes, Ieuan A; Deeb, Asma

    2006-12-01

    Androgen resistance causes the androgen insensitivity syndrome in its variant forms and is a paradigm of clinical syndromes associated with hormone resistance. In its complete form, the syndrome causes XY sex reversal and a female phenotype. Partial resistance to androgens is a common cause of ambiguous genitalia of the newborn, but a similar phenotype may result from several other conditions, including defects in testis determination and androgen biosynthesis. The biological actions of androgens are mediated by a single intracellular androgen receptor encoded by a gene on the long arm of the X chromosome. Mutations in this gene result in varying degrees of androgen receptor dysfunction and phenotypes that often show poor concordance with the genotype. Functional characterization and three-dimensional modelling of novel mutant receptors has been informative in understanding the mechanism of androgen action. Management issues in syndromes of androgen insensitivity include decisions on sex assignment, timing of gonadectomy in relation to tumour risk, and genetic and psychological counselling.

  19. Ankyrin-independent membrane protein-binding sites for brain and erythrocyte spectrin.

    PubMed

    Steiner, J P; Bennett, V

    1988-10-05

    Brain spectrin reassociates in in vitro binding assays with protein(s) in highly extracted brain membranes quantitatively depleted of ankyrin and spectrin. These newly described membrane sites for spectrin are biologically significant and involve a protein since (a) binding occurs optimally at physiological pH (6.7-6.9) and salt concentrations (50 mM), (b) binding is abolished by digestion of membranes with alpha-chymotrypsin, (c) Scatchard analysis is consistent with a binding capacity of at least 50 pmol/mg total membrane protein, and highest affinity of 3 nM. The major ankyrin-independent binding activity of brain spectrin is localized to the beta subunit of spectrin. Brain membranes also contain high affinity binding sites for erythrocyte spectrin, but a 3-4 fold lower capacity than for brain spectrin. Some spectrin-binding sites associate preferentially with brain spectrin, some with erythrocyte spectrin, and some associate with both types of spectrin. Erythrocyte spectrin contains distinct binding domains for ankyrin and brain membrane protein sites, since the Mr = 72,000 spectrin-binding fragment of ankyrin does not compete for binding of spectrin to brain membranes. Spectrin binds to a small number of ankyrin-independent sites in erythrocyte membranes present in about 10,000-15,000 copies/cell or 10% of the number of sites for ankyrin. Brain spectrin binds to these sites better than erythrocyte spectrin suggesting that erythrocytes have residual binding sites for nonerythroid spectrin. Ankyrin-independent-binding proteins that selectively bind to certain isoforms of spectrin provide a potentially important flexibility in cellular localization and time of synthesis of proteins involved in spectrin-membrane interactions. This flexibility has implications for assembly of the membrane skeleton and targeting of spectrin isoforms to specialized regions of cells.

  20. Studies of an Androgen-Binding Protein Knockout Corroborate a Role for Salivary ABP in Mouse Communication.

    PubMed

    Chung, Amanda G; Belone, Phillip M; Vošlajerová Bímová, Barbora; Karn, Robert M; Laukaitis, Christina M

    2017-02-03

    The house mouse Androgen-binding protein (Abp) gene family is comprised of 64 paralogs, 30 Abpa and 34 Abpbg, encoding the alpha (ABPA) and betagamma (ABPBG) protein subunits that are disulfide-bridged to form dimers in secretions. Only 14 Abp genes are expressed in distinct patterns in the lacrimal (11) and submandibular glands (3). We created a knockout mouse line lacking two of the three genes expressed in submandibular glands, Abpa27 and Abpbg27, by replacing them with the neomycin resistance gene. The knockout genotype (-/-) showed no Abpa27 or Abpbg27 transcripts in submandibular gland cDNA libraries and there was a concomitant lack of protein expression of ABPA27 and ABPBG27 in the -/- genotype saliva, shown by elimination of these two proteins from the saliva proteome and the loss of cross-reactive material in the acinar cells of the submandibular glands. We also observed a decrease in BG26 protein in the -/- animals, suggesting monomer instability. Overall, we observed no major phenotypic changes in the -/- genotype, compared with their +/+ and +/- siblings raised in a laboratory setting, including normal growth curves, tissue histology, fecundity and longevity. The only difference is that male and female C57BL/6 mice preferred saliva of the opposite sex containing ABP statistically significantly more than saliva of the opposite sex without ABP in a Y-maze test. These results show for the first time that mice can sense the presence of ABP between saliva targets with and without ABPs, and that they spend more time investigating the target containing ABP.

  1. Characterization of two forms of mouse salivary androgen-binding protein (ABP): implications for evolutionary relationships and ligand-binding function.

    PubMed

    Karn, Robert C; Laukaitis, Christina M

    2003-06-17

    Mouse salivary androgen-binding protein (ABP) is a member of the secretoglobin family produced in the submaxillary glands of house mice (Mus musculus). We report the cDNA sequences and amino acid sequences of the beta and gamma subunits of ABP from a mouse cDNA library, identifying the two subunits by their pIs and molecular weights. An anomalously high molecular weight of the alpha subunit is likely due to glycosylation at a single site. A phylogenetic comparison of the three subunits of ABP with the chains of other mammalian secretoglobins shows that ABP is most closely related to mouse lachrymal protein and to the major cat allergen Fel dI. An evaluation of the most conserved residues in ABP and the other secretoglobins, in light of structural data reported by others [Callebaut, I., Poupon, A., Bally, R., Demaret, J.-P., Housset, D., Delettre, J., Hossenlopp, P., and Mornon, J.-P. (2000) Ann. N.Y. Acad. Sci. 923, 90-112; Pattabiraman, N., Matthews, J., Ward, K., Mantile-Selvaggi, G., Miele, L., and Mukherjee, A. (2000) Ann. N.Y. Acad. Sci. 923, 113-127], allows us to draw conclusions about the critical residues important in ligand binding by the two different ABP dimers and to assess the importance of ligand binding in the function of the molecule. In addition to the cDNAs, which represent those of the musculus subspecies of Mus musculus, we also report the coding regions of the beta and gamma subunit cDNAs from two other mouse inbred strains which represent the other two subspecies: M. musculus domesticus and M. musculus castaneus. The high nonsynonymous/synonymous substitution rate ratios (K(a)/K(s)) for both the beta and gamma subunits suggest that these two proteins are evolving under strong directional selection, as has been reported for the alpha subunit [Hwang, J., Hofstetter, J., Bonhomme, F., and Karn, R. (1997) J. Hered. 88, 93-97; Karn, R., and Clements, M. (1999) Biochem. Genet. 37, 187-199].

  2. Tight binding of NAP-22 with acidic membrane lipids.

    PubMed

    Maekawa, Shohei; Kobayashi, Yuumi; Morita, Mitsuhiro; Suzaki, Toshinobu

    2015-07-23

    Recovery of various signal transduction molecules in the detergent-resistant membrane microdomain (DRM) fraction suggests the importance of this region in cellular functions. Insolubility of the outer leaflet of DRM to the non-ionic detergent is ascribed to the tight association of cholesterol and sphingolipid. Since, poor localization of sphingolipid is observed in the inner leaflet, the physicochemical background of the insolubility of the inner leaflet is hence still an enigma. NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched calmodulin-binding protein and one of the major proteins in the DRM of the neuronal cell membrane. A previous study showed the presence of several lipids in a NAP-22 fraction after the process of extraction and column chromatography. In this study, the effect of lipid extraction on NAP-22 was studied through native-gel electrophoresis, ultracentrifugation, and electron microscopic observation. The mobility of NAP-22 in native-PAGE was shifted from low to high after delipidation. Delipidated NAP-22 bound phosphatidylserine (PS), phosphatidylinosotol, and ganglioside. Some part of the mixture of PS and NAP-22 was recovered in the insoluble fraction after Triton X-100 treatment and the addition of cholesterol enhanced the amount of NAP-22 in the insoluble fraction.

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

    PubMed

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

    2015-12-28

    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.

  4. Repeated anabolic-androgenic steroid treatment during adolescence increases vasopressin V(1A) receptor binding in Syrian hamsters: correlation with offensive aggression.

    PubMed

    DeLeon, Katrina R; Grimes, Jill M; Melloni, Richard H

    2002-09-01

    Repeated anabolic-androgenic steroid treatment during adolescence increases hypothalamic vasopressin and facilitates offensive aggression in male Syrian hamsters (Mesocricetus auratus). The current study investigated whether anabolic-androgenic steroid exposure during this developmental period influenced vasopressin V(1A) receptor binding activity in the hypothalamus and several other brain areas implicated in aggressive behavior in hamsters. To test this, adolescent male hamsters were administered anabolic steroids or sesame oil throughout adolescence, tested for offensive aggression, and examined for differences in vasopressin V(1A) receptor binding using in situ autoradiography. When compared with control animals, aggressive, adolescent anabolic steroid-treated hamsters showed significant increases (20-200%) in the intensity of vasopressin V(1A) receptor labeling in several aggression areas, including the ventrolateral hypothalamus, bed nucleus of the stria terminalis, and lateral septum. However, no significant differences in vasopressin V(1A) receptor labeling were found in other brain regions implicated in aggressive responding, most notably the lateral zone from the medial preoptic area to anterior hypothalamus and the corticomedial amygdala. These data suggest that adolescent anabolic steroid exposure may facilitate offensive aggression by increasing vasopressin V(1A) receptor binding in several key areas of the hamster brain.

  5. 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

  6. Cell membrane chromatography competitive binding analysis for characterization of α1A adrenoreceptor binding interactions.

    PubMed

    Du, Hui; Ren, Jing; Wang, Sicen; He, Langchong

    2011-07-01

    A new high α(1A) adrenoreceptor (α(1A)AR) expression cell membrane chromatography (CMC) method was developed for characterization of α(1A)AR binding interactions. HEK293 α(1A) cell line, which expresses stably high levels of α(1A)AR, was used to prepare the stationary phase in the CMC model. The HEK293 α(1A)/CMC-offline-HPLC system was applied to specifically recognize the ligands which interact with the α(1A)AR, and the dissociation equilibrium constants (K (D)) obtained from the model were (1.87 ± 0.13) × 10(-6) M for tamsulosin, (2.86 ± 0.20) × 10(-6) M for 5-methylurapidil, (3.01 ± 0.19) × 10(-6) M for doxazosin, (3.44 ± 0.19) × 10(-6) M for terazosin, (3.50 ± 0.21) × 10(-6) M for alfuzosin, and (7.57 ± 0.31) × 10(-6) M for phentolamine, respectively. The competitive binding study between tamsulosin and terazosin indicated that the two drugs interacted at the common binding site of α(1A)AR. However, that was not the case between tamsulosin and oxymetazoline. The results had a positive correlation with those from radioligand binding assay and indicated that the CMC method combined modified competitive binding could be a quick and efficient way for characterizing the drug-receptor interactions.

  7. 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.

  8. The HOPS/class C Vps complex tethers membranes by binding to one Rab GTPase in each apposed membrane.

    PubMed

    Ho, Ruoya; Stroupe, Christopher

    2015-07-15

    Many Rab GTPase effectors are membrane-tethering factors, that is, they physically link two apposed membranes before intracellular membrane fusion. In this study, we investigate the distinct binding factors needed on apposed membranes for Rab effector-dependent tethering. We show that the homotypic fusion and protein-sorting/class C vacuole protein-sorting (HOPS/class C Vps) complex can tether low-curvature membranes, that is, liposomes with a diameter of ∼100 nm, only when the yeast vacuolar Rab GTPase Ypt7p is present in both tethered membranes. When HOPS is phosphorylated by the vacuolar casein kinase I, Yck3p, tethering only takes place when GTP-bound Ypt7p is present in both tethered membranes. When HOPS is not phosphorylated, however, its tethering activity shows little specificity for the nucleotide-binding state of Ypt7p. These results suggest a model for HOPS-mediated tethering in which HOPS tethers membranes by binding to Ypt7p in each of the two tethered membranes. Moreover, because vacuole-associated HOPS is presumably phosphorylated by Yck3p, our results suggest that nucleotide exchange of Ypt7p on multivesicular bodies (MVBs)/late endosomes must take place before HOPS can mediate tethering at vacuoles.

  9. Membrane-Protein Binding Measured with Solution-Phase Plasmonic Nanocube Sensors

    PubMed Central

    Wu, Hung-Jen; Henzie, Joel; Lin, Wan-Chen; Rhodes, Christopher; Li, Zhu; Sartorel, Elodie; Thorner, Jeremy; Yang, Peidong; Groves, Jay. T.

    2013-01-01

    We describe a solution-phase sensor of lipid-protein binding based on localized surface plasmon resonance (LSPR) of silver nanocubes. When silica-coated nanocubes are mixed into a suspension of lipid vesicles, supported membranes spontaneously assemble on their surfaces. Using a standard laboratory spectrophotometer, we calibrate the LSPR peak shift due to protein binding to the membrane surface and then characterize the lipid-binding specificity of a pleckstrin-homology domain protein. PMID:23085614

  10. An Experimentally Based Computer Search Identifies Unstructured Membrane-binding Sites in Proteins

    PubMed Central

    Brzeska, Hanna; Guag, Jake; Remmert, Kirsten; Chacko, Susan; Korn, Edward D.

    2010-01-01

    Programs exist for searching protein sequences for potential membrane-penetrating segments (hydrophobic regions) and for lipid-binding sites with highly defined tertiary structures, such as PH, FERM, C2, ENTH, and other domains. However, a rapidly growing number of membrane-associated proteins (including cytoskeletal proteins, kinases, GTP-binding proteins, and their effectors) bind lipids through less structured regions. Here, we describe the development and testing of a simple computer search program that identifies unstructured potential membrane-binding sites. Initially, we found that both basic and hydrophobic amino acids, irrespective of sequence, contribute to the binding to acidic phospholipid vesicles of synthetic peptides that correspond to the putative membrane-binding domains of Acanthamoeba class I myosins. Based on these results, we modified a hydrophobicity scale giving Arg- and Lys-positive, rather than negative, values. Using this basic and hydrophobic scale with a standard search algorithm, we successfully identified previously determined unstructured membrane-binding sites in all 16 proteins tested. Importantly, basic and hydrophobic searches identified previously unknown potential membrane-binding sites in class I myosins, PAKs and CARMIL (capping protein, Arp2/3, myosin I linker; a membrane-associated cytoskeletal scaffold protein), and synthetic peptides and protein domains containing these newly identified sites bound to acidic phospholipids in vitro. PMID:20018884

  11. Measurement of the binding parameters of annexin derivative-erythrocyte membrane interactions.

    PubMed

    Yen, Tzu-Chen; Wey, Shiaw-Pyng; Liao, Chang-Hui; Yeh, Chi-Hsiao; Shen, Duan-Wen; Achilefu, Samuel; Wun, Tze-Chein

    2010-11-01

    Erythrocyte ghosts prepared from fresh blood expressed phosphatidylserine (PS) on the membrane surfaces in a rather stable fashion. The binding of fluorescein-5-isothiocyanate (FITC)-labeled annexin V (ANV) derivatives to these membranes was studied by titration with proteins and with calcium. Whereas the preaddition of ethylenediaminetetraacetic acid (EDTA) to reaction mixtures totally prevented membrane binding, Ca(2+)-dependent binding was only partially reversed by EDTA treatment, consistent with an initial Ca(2+)-dependent binding that became partially Ca(2+) independent. Data derived from saturation titration with ANV derivatives poorly fit the simple protein-membrane equilibrium binding equation and showed negative cooperativity of binding with increasing membrane occupancy. In contrast, calcium titration at low binding site occupancy resulted in excellent fit into the protein-Ca(2+)-membrane equilibrium binding equation. Calcium titrations of FITC-labeled ANV and ANV-6L15 (a novel ANV-Kunitz protease inhibitor fusion protein) yielded a Hill coefficient of approximately 4 in both cases. The apparent dissociation constant for ANV-6L15 was approximately 4-fold lower than that of ANV at 1.2-2.5mM Ca(2+). We propose that ANV-6L15 may provide improved detection of PS exposed on the membrane surfaces of pathological cells in vitro and in vivo.

  12. In Vitro Auxin Binding to Cellular Membranes of Cucumber Fruits 123

    PubMed Central

    Narayanan, Komaratchi R.; Mudge, Kenneth W.; Poovaiah, B. W.

    1981-01-01

    Specific binding of 1-naphthaleneacetic acid (NAA) to crude membrane preparations from cucumber (Cucumis sativus L.) was demonstrated. This in vitro binding had a pH optimum of 3.75 and an equilibrium dissociation constant of 10 to 20 micromolar with 1250 picomoles binding sites per gram fresh weight. The NAA-binding sites were pronase sensitive. The supernatant from the fruit partially inhibited the in vitro NAA binding to fruit membranes. NAA, 2-naphthoxyacetic acid, 3-indoleacetic acid, 2-4-dichlorophenoxyacetic acid, and 2,3,5-triiodobenzoic acid, which are reported to be very good inducers of parthenocarpy in cucumber, showed a high degree of specific binding to cucumber fruit membranes. In comparison, 2-naphthaleneacetic acid and indolepropionic acid, which are reported to be very weak auxins in corn coleoptile, pea stem, and strawberry fruit growth bioassays, did not bind efficiently to cucumber fruit membranes. In vitro binding studies with fruit membranes suggest that auxin stimulated fruit growth may be mediated by membrane-associated, auxin-binding protein(s). PMID:16661764

  13. A G577R mutation in the human AR P box results in selective decreases in DNA binding and in partial androgen insensitivity syndrome.

    PubMed

    Nguyen, D; Steinberg, S V; Rouault, E; Chagnon, S; Gottlieb, B; Pinsky, L; Trifiro, M; Mader, S

    2001-10-01

    We have characterized a novel mutation of the human AR, G577R, associated with partial androgen insensitivity syndrome. G577 is the first amino acid of the P box, a region crucial for the selectivity of receptor/DNA interaction. Although the equivalent amino acid in the GR (also Gly) is not involved in DNA interaction, the residue at the same position in the ER (Glu) interacts with the two central base pairs in the PuGGTCA motif. Using a panel of 16 palindromic probes that differ in these base pairs (PuGNNCA) in gel shift experiments with either the AR DNA-binding domain or the full length receptor, we observed that the G577R mutation does not induce binding to probes that are not recognized by the wild-type AR. However, binding to the four PuGNACA elements recognized by the wild-type AR was affected to different degrees, resulting in an altered selectivity of DNA response element recognition. In particular, AR-G577R did not interact with PuGGACA palindromes. Modeling of the complex between mutant AR and PuGNACA motifs indicates that the destabilizing effect of the mutation is attributable to a steric clash between the C beta of Arg at position 1 of the P box and the methyl group of the second thymine residue in the TGTTCPy arm of the palindrome. In addition, the Arg side chain can interact with G or T at the next position (PuGCACA and PuGAACA elements, respectively). The presence of C is not favorable, however, because of incompatible charges, abrogating binding to the PuGGACA element. Transactivation of several natural or synthetic promoters containing PuGGACA motifs was drastically reduced by the G577R mutation. These data suggest that androgen target genes may be differentially affected by the G577R mutation, the first natural mutation characterized that alters the selectivity of the AR/DNA interaction. This type of mutation may thus contribute to the diversity of phenotypes associated with partial androgen insensitivity syndrome.

  14. Characterization of protein-membrane binding interactions via a microplate assay employing whole liposome immobilization.

    PubMed

    Smith, Matthew D; Best, Michael D

    2011-01-01

    Protein-cell membrane binding interactions control numerous vital biological processes, many of which can go awry during disease onset. However, the study of these events is complicated by the complexity of the membrane bilayer. These efforts would benefit from a rapid and easily accessible method for characterizing protein-membrane recognition events. Herein, we describe a microplate-based method for the detection of protein-membrane binding that employs whole liposome immobilization using a biotin anchor. First, control studies are detailed to test for nonspecific liposome immobilization (fluorescence assay; see Subheading 3.2), and to ensure that liposomes remain intact on the microplate surface (dye leakage assay; see Subheading 3.3). Finally, a protein-membrane binding detection assay is described through the example of protein kinase Cα binding to surface-immobilized whole liposomes (see Subheading 3.4).

  15. DJBP: a novel DJ-1-binding protein, negatively regulates the androgen receptor by recruiting histone deacetylase complex, and DJ-1 antagonizes this inhibition by abrogation of this complex.

    PubMed

    Niki, Takeshi; Takahashi-Niki, Kazuko; Taira, Takahiro; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2003-02-01

    DJ-1 was identified by us as a novel oncogene that transforms mouse NIH3T3 cells in cooperation with ras. We later identified PIAS (protein inhibitor of activated STAT)xalpha as a DJ-1-binding protein, and found that DJ-1 restored androgen receptor (AR) transcription activity that was repressed by PIASxalpha. To further characterize the function of DJ-1, we cloned cDNA encoding a novel DJ-1-binding protein, DJBP, by a yeast two-hybrid system. DJBP mRNA was found to be specifically expressed in the testis. In addition to the binding of DJBP to the COOH-terminal region of DJ-1, DJBP was also found to bind in vitro and in vivo to the DNA-binding domain of the AR in a testosterone-dependent manner and to be colocalized with DJ-1 or AR in the nucleus. Furthermore, a co-immunoprecipitation assay showed that the formation of a ternary complex between DJ-1, DJBP, and AR occurred in cells in which DJ-1 bound to the AR via DJBP. It was found that DJBP repressed a testosterone-dependent AR transactivation activity in monkey Cos1 cells by recruiting histone deacetylase (HDAC) complex, including HDAC1 and mSin3, and that DJ-1 partially restored its repressed activity by abrogating DJBP-HDAC complex. These results suggest that AR is positively regulated by DJ-1, which antagonizes the function of negative regulators, including DJBP.

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

    PubMed

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

    2016-04-06

    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.

  17. Entropic and enthalpic contributions to annexin V-membrane binding: a comprehensive quantitative model.

    PubMed

    Jeppesen, Brian; Smith, Christina; Gibson, Donald F; Tait, Jonathan F

    2008-03-07

    Annexin V binds to membranes with very high affinity, but the factors responsible remain to be quantitatively elucidated. Analysis by isothermal microcalorimetry and calcium titration under conditions of low membrane occupancy showed that there was a strongly positive entropy change upon binding. For vesicles containing 25% phosphatidylserine at 0.15 m ionic strength, the free energy of binding was -53 kcal/mol protein, whereas the enthalpy of binding was -38 kcal/mol. Addition of 4 m urea decreased the free energy of binding by about 30% without denaturing the protein, suggesting that hydrophobic forces make a significant contribution to binding affinity. This was confirmed by mutagenesis studies that showed that binding affinity was modulated by the hydrophobicity of surface residues that are likely to enter the interfacial region upon protein-membrane binding. The change in free energy was quantitatively consistent with predictions from the Wimley-White scale of interfacial hydrophobicity. In contrast, binding affinity was not increased by making the protein surface more positively charged, nor decreased by making it more negatively charged, ruling out general ionic interactions as major contributors to binding affinity. The affinity of annexin V was the same regardless of the head group present on the anionic phospholipids tested (phosphatidylserine, phosphatidylglycerol, phosphatidylmethanol, and cardiolipin), ruling out specific interactions between the protein and non-phosphate moieties of the head group as a significant contributor to binding affinity. Analysis by fluorescence resonance energy transfer showed that multimers did not form on phosphatidylserine membranes at low occupancy, indicating that annexin-annexin interactions did not contribute to binding affinity. In summary, binding of annexin V to membranes is driven by both enthalpic and entropic forces. Dehydration of hydrophobic regions of the protein surface as they enter the interfacial region

  18. Characterization of Cytokinetic F-BARs and Other Membrane-Binding Proteins.

    PubMed

    McDonald, Nathan A; Gould, Kathleen L

    2016-01-01

    Multiple membrane-binding proteins are key players in cytokinesis in yeast and other organisms. In vivo techniques for analyzing protein-membrane interactions are currently limited. In vitro assays allow characterization of the biochemical properties of these proteins to build a mechanistic understanding of protein-membrane interactions during cytokinesis. Here, we describe two in vitro assays to characterize FCH-Bin/Amphyphysin/RVS (F-BAR) domains and other protein's interactions with membranes: liposome co-pelleting and giant unilamellar vesicle fluorescent binding.

  19. Silibinin inhibits aberrant lipid metabolism, proliferation and emergence of androgen-independence in prostate cancer cells via primarily targeting the sterol response element binding protein 1.

    PubMed

    Nambiar, Dhanya K; Deep, Gagan; Singh, Rana P; Agarwal, Chapla; Agarwal, Rajesh

    2014-10-30

    Prostate cancer (PCA) kills thousands of men every year, demanding additional approaches to better understand and target this malignancy. Recently, critical role of aberrant lipogenesis is highlighted in prostate carcinogenesis, offering a unique opportunity to target it to reduce PCA. Here, we evaluated efficacy and associated mechanisms of silibinin in inhibiting lipid metabolism in PCA cells. At physiologically achievable levels in human, silibinin strongly reduced lipid and cholesterol accumulation specifically in human PCA cells but not in non-neoplastic prostate epithelial PWR-1E cells. Silibinin also decreased nuclear protein levels of sterol regulatory element binding protein 1 and 2 (SREBP1/2) and their target genes only in PCA cells. Mechanistically, silibinin activated AMPK, thereby increasing SREBP1 phosphorylation and inhibiting its nuclear translocation; AMPK inhibition reversed silibinin-mediated decrease in nuclear SREBP1 and lipid accumulation. Additionally, specific SREBP inhibitor fatostatin and stable overexpression of SREBP1 further confirmed the central role of SREBP1 in silibinin-mediated inhibition of PCA cell proliferation and lipid accumulation and cell cycle arrest. Importantly, silibinin also inhibited synthetic androgen R1881-induced lipid accumulation and completely abrogated the development of androgen-independent LNCaP cell clones via targeting SREBP1/2. Together, these mechanistic studies suggest that silibinin would be effective against PCA by targeting critical aberrant lipogenesis.

  20. Cell Migration and Invadopodia Formation Require a Membrane-binding Domain of CARMIL2.

    PubMed

    Lanier, M Hunter; McConnell, Patrick; Cooper, John A

    2016-01-15

    CARMILs regulate capping protein (CP), a critical determinant of actin assembly and actin-based cell motility. Vertebrates have three conserved CARMIL genes with distinct functions. In migrating cells, CARMIL2 is important for cell polarity, lamellipodial assembly, ruffling, and macropinocytosis. In cells, CARMIL2 localizes with a distinctive dual pattern to vimentin intermediate filaments and to membranes at leading edges and macropinosomes. The mechanism by which CARMIL2 localizes to membranes has not been defined. Here, we report that CARMIL2 has a conserved membrane-binding domain composed of basic and hydrophobic residues, which is necessary and sufficient for membrane localization, based on expression studies in cells and on direct binding of purified protein to lipids. Most important, we find that the membrane-binding domain is necessary for CARMIL2 to function in cells, based on rescue expression with a set of biochemically defined mutants. CARMIL1 and CARMIL3 contain similar membrane-binding domains, based on sequence analysis and on experiments, but other CPI motif proteins, such as CD2AP, do not. Based on these results, we propose a model in which the membrane-binding domain of CARMIL2 tethers this multidomain protein to the membrane, where it links dynamic vimentin filaments with regulation of actin assembly via CP.

  1. Cell Migration and Invadopodia Formation Require a Membrane-binding Domain of CARMIL2*

    PubMed Central

    Lanier, M. Hunter; McConnell, Patrick; Cooper, John A.

    2016-01-01

    CARMILs regulate capping protein (CP), a critical determinant of actin assembly and actin-based cell motility. Vertebrates have three conserved CARMIL genes with distinct functions. In migrating cells, CARMIL2 is important for cell polarity, lamellipodial assembly, ruffling, and macropinocytosis. In cells, CARMIL2 localizes with a distinctive dual pattern to vimentin intermediate filaments and to membranes at leading edges and macropinosomes. The mechanism by which CARMIL2 localizes to membranes has not been defined. Here, we report that CARMIL2 has a conserved membrane-binding domain composed of basic and hydrophobic residues, which is necessary and sufficient for membrane localization, based on expression studies in cells and on direct binding of purified protein to lipids. Most important, we find that the membrane-binding domain is necessary for CARMIL2 to function in cells, based on rescue expression with a set of biochemically defined mutants. CARMIL1 and CARMIL3 contain similar membrane-binding domains, based on sequence analysis and on experiments, but other CPI motif proteins, such as CD2AP, do not. Based on these results, we propose a model in which the membrane-binding domain of CARMIL2 tethers this multidomain protein to the membrane, where it links dynamic vimentin filaments with regulation of actin assembly via CP. PMID:26578515

  2. Tau binds to lipid membrane surfaces via short amphipathic helices located in its microtubule-binding repeats.

    PubMed

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

    2014-09-16

    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 insights

  3. 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

  4. 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.

  5. Interaction of serum sex steroid-binding globulin with cell membranes of human decidual tissue

    SciTech Connect

    Avvakumov, G.V.; Survilo, L.I.; Strel'chenok, O.A.

    1986-01-20

    The interaction of the sex steroid-binding globulin (SBG) of human blood with plasma membranes of cells from human decidual tissue - the target tissue of estradiol - was studied. It was shown that SBG in complex with estradiol is capable of interacting specifically with these membranes. The dissociation (K/sub dis/) of this interaction is equal to (3.5 +/- 2.0) 10/sup -12/ M. The interaction of the SBG-estradiol complex with the membranes is characterized by high selectivity: such blood serum globulins as albumin, orosomucoid, transferrin, transcortin, and thyroxine-binding globulin do not compete with SBG for its binding sites on the membranes. The SBG-testosterone complex and SBG without steroid are also incapable of interacting with the membranes.

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

    PubMed Central

    Asencio-Hernández, Julia; Kieffer, Bruno

    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

  7. Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

    PubMed Central

    Barros, Marilia; Nanda, Hirsh

    2016-01-01

    ABSTRACT By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals—electrostatic, hydrophobic, and lipid-specific—to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane

  8. Mechanisms of Membrane Binding of Small GTPase K-Ras4B Farnesylated Hypervariable Region*

    PubMed Central

    Jang, Hyunbum; Abraham, Sherwin J.; Chavan, Tanmay S.; Hitchinson, Ben; Khavrutskii, Lyuba; Tarasova, Nadya I.; Nussinov, Ruth; Gaponenko, Vadim

    2015-01-01

    K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. PMID:25713064

  9. Characterization of antibody binding to cell surface antigens using a plasma membrane-bound plate assay.

    PubMed

    Vater, C A; Reid, K; Bartle, L M; Goldmacher, V S

    1995-01-01

    A procedure has been developed for measuring antibody binding to cell surface antigens using an immobilized plasma membrane fraction. In this method, isolated plasma membranes are dried onto wells of a 96-well microtiter plate and incubated with antibodies that recognize a cell surface protein. Bound antibody is detected indirectly using an enzyme-linked or fluorescently tagged second antibody. Alternatively, the primary antibody itself can be labeled and its binding can be detected directly. The assay is simple and fast and provides several advantages over whole cell binding assays currently in widespread use.

  10. Insights into binding of cholera toxin to GM1 containing membrane.

    PubMed

    Basu, Ipsita; Mukhopadhyay, Chaitali

    2014-12-23

    Interactions of cholera toxin (CT) with membrane are associated with the massive secretory diarrhea seen in Asiatic cholera. Ganglioside GM1 has been shown to be responsible for the binding of the B subunit of cholera toxin (CT-B), which then helps CT to pass through the membrane, but the exact mechanism remains to be explored. In this work, we have carried out atomistic scale molecular dynamics simulation to investigate the structural changes of CT upon membrane binding and alteration in membrane structure and dynamics. Starting from the initial structure where the five units of B subunit bind with five GM1, only three of five units remain bound and the whole CT is tilted such that the three binding units are deeper in the membrane. The lipids that are in contact with those units of the CT-B behave differently from the rest of the lipids. Altogether, our results demonstrate the atomistic interaction of CT with GM1 containing lipid membrane and provide a probable mechanism of the early stage alteration of lipid structure and dynamics, which can make a passage for penetration of CT on membrane surface.

  11. Specific binding of toxin II from Centruroides suffusus suffusus to the sodium channel in electroplaque membranes.

    PubMed

    Wheeler, K P; Barhanin, J; Lazdunski, M

    1982-10-26

    The binding of toxin II from the scorpion Centruroides suffusus suffusus (CssII) to electroplaque membranes from Electrophorus electricus was studied with the use of a radiolabeled derivative of the toxin ([125I]CssII). Specific binding of the latter to the membranes required the protonation of a group, either in the membrane or in the toxin itself, with an apparent pKa value of 7.5 and also the presence of a certain minimum concentration of ions, though there was no requirement for a specific ion. At 20 degrees C and pH 6 the second-order rate constant for formation of the [125I]CssII-membrane complex was about 5 X 10(6) M-1 s-1, while the first-order constant for its dissociation was about 2 X 10(-3) s-1. Under equilibrium conditions specific binding of [125I]CssII was a simple saturable function of [125I]CssII concentration, characterized by a dissociation constant of 0.4-0.7 nM and a maximum capacity of 0.9-2.4 pmol of toxin/mg of membrane protein. The latter value was the same as the number of membrane sites that could specifically bind a radiolabeled derivative of tetrodotoxin. Unlabeled CssII displaced bound [125I]CssII with an apparent dissociation constant of about 1 nM. None of 19 other neurotoxins or local anaesthetics known to interact with Na+ channels in excitable cells affected [125I]CssII binding, but it was completely inhibited by toxin gamma from the scorpion Tityus serrulatus serrulatus. These findings suggest that the Na+ channel possesses a distinct class of binding sites to which these two scorpion toxins bind with high affinities. On the other hand, no CssII receptor was detected in crab axonal membranes, indicating that it is not a characteristic feature of all Na+ channels.

  12. 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.

  13. Androgen insensitivity syndrome.

    PubMed

    Hughes, Ieuan Arwel; Werner, Ralf; Bunch, Trevor; Hiort, Olaf

    2012-10-01

    The androgen insensitivity syndromes (AIS) fall within the generic category of 46,XY DSD (disorder of sex development) and present as phenotypes associated with complete or partial resistance to the action of androgens. Three categories are recognized: complete androgen insensitivity syndrome (CAIS), partial androgen insensitivity syndrome (PAIS), mild androgen insensitivity syndrome (MAIS). The androgen receptor (AR) is encoded by an 8 exon gene on the X chromosome long arm. More than 800 mutations in the AR gene have been reported in AIS patients (www.androgendb.mcgill.ca/). They are distributed throughout the gene with a preponderance located in the ligand binding domain. The most severe mutations are generally associated with a CAIS phenotype, but the correlation is less defined in PAIS. CAIS presents typically as primary amenorrhoea in an adolescent female and less commonly in infancy with bilateral inguinal/labial swellings due to testes. The differential diagnosis in CAIS is limited, whereas in PAIS, numerous other causes of DSD can also produce the typical phenotype of micropenis, severe hypospadias and bifid scrotum. Management issues in CAIS involve timing of gonadectomy, appropriate hormone replacement therapy and assessment of the need for vaginal dilation or rarely, vaginal surgery. The risk of gonadal germ cell tumor is low during childhood and adolescence but increases in later adulthood. Expert psychological counseling is mandatory to manage the disconnect between chromosomal, gonadal and phenotypic sex and to choreograph the evolving process of disclosure from late childhood through to maturity. It is implicit that management in AIS requires a multidisciplinary team and engagement with patient advocacy groups.

  14. Sintokamide A Is a Novel Antagonist of Androgen Receptor That Uniquely Binds Activation Function-1 in Its Amino-terminal Domain*

    PubMed Central

    Banuelos, Carmen A.; Tavakoli, Iran; Tien, Amy H.; Caley, Daniel P.; Mawji, Nasrin R.; Li, Zhenzhen; Wang, Jun; Yang, Yu Chi; Imamura, Yusuke; Yan, Luping; Wen, Jian Guo; Andersen, Raymond J.; Sadar, Marianne D.

    2016-01-01

    Androgen receptor (AR) is a validated drug target for all stages of prostate cancer including metastatic castration-resistant prostate cancer (CRPC). All current hormone therapies for CRPC target the C-terminal ligand-binding domain of AR and ultimately all fail with resumed AR transcriptional activity. Within the AR N-terminal domain (NTD) is activation function-1 (AF-1) that is essential for AR transcriptional activity. Inhibitors of AR AF-1 would potentially block most AR mechanisms of resistance including constitutively active AR splice variants that lack the ligand-binding domain. Here we provide evidence that sintokamide A (SINT1) binds AR AF-1 region to specifically inhibit transactivation of AR NTD. Consistent with SINT1 targeting AR AF-1, it attenuated transcriptional activities of both full-length AR and constitutively active AR splice variants, which correlated with inhibition of growth of enzalutamide-resistant prostate cancer cells expressing AR splice variants. In vivo, SINT1 caused regression of CRPC xenografts and reduced expression of prostate-specific antigen, a gene transcriptionally regulated by AR. Inhibition of AR activity by SINT1 was additive to EPI-002, a known AR AF-1 inhibitor that is in clinical trials (NCT02606123). This implies that SINT1 binds to a site on AF-1 that is unique from EPI. Consistent with this suggestion, these two compounds showed differences in blocking AR interaction with STAT3. This work provides evidence that the intrinsically disordered NTD of AR is druggable and that SINT1 analogs may provide a novel scaffold for drug development for the treatment of prostate cancer or other diseases of the AR axis. PMID:27576691

  15. 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

  16. Novel affinity membranes with macrocyclic spacer arms synthesized via click chemistry for lysozyme binding.

    PubMed

    Lin, Ligang; Sun, Hui; Zhang, Kaiyu; Zhong, Yonghui; Cheng, Qi; Bian, Xihui; Xin, Qingping; Cheng, Bowen; Feng, Xianshe; Zhang, Yuzhong

    2017-04-05

    Affinity membrane has great potential for applications in bioseparation and purification. Disclosed herein is the design of a novel affinity membrane with macrocyclic spacer arms for lysozyme binding. The clickable azide-cyclodextrin (CD) arms and clickable alkyne ethylene-vinyl alcohol (EVAL) chains are designed and prepared. By the azide-alkyne click reaction, the EVAL-CD-ligands affinity membranes with CD spacer arms in three-dimensional micro channels have been successfully fabricated. The FT-IR, XPS, NMR, SEM and SEM-EDS results give detailed information of structure evolution. The abundant pores in membrane matrix provide efficient working channels, and the introduced CD arms with ligands (affinity sites) provide supramolecular atmosphere. Compared with that of raw EVAL membrane, the adsorption capacity of EVAL-CD-ligands membrane (26.24mg/g) show a triple increase. The study indicates that three effects (inducing effect, arm effect, site effect) from CD arms render the enhanced performance. The click reaction happened in membrane matrix in bulk. The effective lysozyme binding and higher adsorption performance of affinity membranes described herein compared with other reported membranes are markedly related with the proposed strategy involving macrocyclic spacer arms and supramolecular working channels.

  17. 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.

  18. Modulation of function in a minimalist heme-binding membrane protein.

    PubMed

    Shinde, Sandip; Cordova, Jeanine M; Woodrum, Brian W; Ghirlanda, Giovanna

    2012-04-01

    De novo designed heme-binding proteins have been used successfully to recapitulate features of natural hemoproteins. This approach has now been extended to membrane-soluble model proteins. Our group designed a functional hemoprotein, ME1, by engineering a bishistidine binding site into a natural membrane protein, glycophorin A (Cordova et al. in J Am Chem Soc 129:512-518, 2007). ME1 binds iron(III) protoporphyrin IX with submicromolar affinity, has a redox potential of -128 mV, and displays peroxidase activity. Here, we show the effect of aromatic residues in modulating the redox potential in the context of a membrane-soluble model system. We designed aromatic interactions with the heme through a single-point mutant, G25F, in which a phenylalanine is designed to dock against the porphyrin ring. This mutation results in roughly tenfold tighter binding to iron(III) protoporphyrin IX (K(d,app) = 6.5 × 10(-8) M), and lowers the redox potential of the cofactor to -172 mV. This work demonstrates that specific design features aimed at controlling the properties of bound cofactors can be introduced in a minimalist membrane hemoprotein model. The ability to modulate the redox potential of cofactors embedded in artificial membrane proteins is crucial for the design of electron transfer chains across membranes in functional photosynthetic devices.

  19. Membrane Binding by CHMP7 Coordinates ESCRT-III-Dependent Nuclear Envelope Reformation.

    PubMed

    Olmos, Yolanda; Perdrix-Rosell, Anna; Carlton, Jeremy G

    2016-10-10

    In addition to its role in membrane abscission during cytokinesis, viral budding, endosomal sorting, and plasma membrane repair [1], the endosomal sorting complex required for transport-III (ESCRT-III) machinery has recently been shown to seal holes in the reforming nuclear envelope (NE) during mitotic exit [2, 3]. ESCRT-III also acts during interphase to repair the NE upon migration-induced rupture [4, 5], highlighting its key role as an orchestrator of membrane integrity at this organelle. While NE localization of ESCRT-III is dependent upon the ESCRT-III component CHMP7 [3], it is unclear how this complex is able to engage nuclear membranes. Here we show that the N terminus of CHMP7 acts as a novel membrane-binding module. This membrane-binding ability allows CHMP7 to bind to the ER, an organelle continuous with the NE, and it provides a platform to direct NE recruitment of ESCRT-III during mitotic exit. CHMP7's N terminus comprises tandem Winged-Helix domains [6], and, by using homology modeling and structure-function analysis, we identify point mutations that disrupt membrane binding and prevent both ER localization of CHMP7 and its subsequent enrichment at the reforming NE. These mutations also prevent assembly of downstream ESCRT-III components at the reforming NE and proper establishment of post-mitotic nucleo-cytoplasmic compartmentalization. These data identify a novel membrane-binding activity within an ESCRT-III subunit that is essential for post-mitotic nuclear regeneration.

  20. Thermodynamics of RTA3 peptide binding to membranes and consequences for antimicrobial activity☆

    PubMed Central

    Hawrani, Ayman; Howe, Robin A.; Walsh, Timothy R.; Dempsey, Christopher E.

    2010-01-01

    RTA3 is an α-helical, amphipathic peptide with broad-spectrum activity against Gram-negative bacteria and low mammalian cell toxicity. RTA3 contains a cysteine residue, replacement of which with an alanine or serine (RTA3-C15S) virtually abolishes antimicrobial activity. Much of the activity of RTA3 can be recovered in RTA3-C15L, indicating that the C15 residue functions largely as a bulky hydrophobic side chain promoting target cell membrane interactions. The poorly active RTA3-C15S is a useful variant for assessing the mechanistic aspects of RTA3 activity. Binding and membrane perturbation in vesicles containing different proportions of negative surface charge are analyzed in terms of amino acid-specific free energy contributions to interfacial binding, which likely underlie variations in antimicrobial activity amongst RTA3 variants. Comparison with published free energy scales indicates that the reduced electrostatic contribution to binding to membranes having reduced negative surface charge can be compensated in RTA3 (but not RTA3-C15S) by a slightly deeper insertion of the C-terminus of the peptide to maximize hydrophobic contributions to binding. Analysis of inner membrane (IM)- and outer membrane (OM)-selective permeabilization of Escherichiacoli demonstrates a broad similarity between peptide effects on vesicles with low negative surface charge (20% negatively charged lipids), E.coli membrane perturbation, and antimicrobial activity, supporting a role for membrane perturbation in the killing mechanism of RTA3. The results demonstrate that large variations in antimicrobial activity on subtle changes in amino acid sequence in helical amphipathic peptides can be rationalized in terms of the thermodynamics of peptide binding to membranes, allowing a more systematic understanding of antimicrobial activity in these peptides. PMID:20346912

  1. Binding of Cerebratulus cytolysin A-III to human erythrocyte membranes.

    PubMed

    Blumenthal, K M

    1985-01-10

    Binding of Cerebratulus lacteus cytolysin A-III to intact human erythrocytes and erythrocyte membranes has been investigated. Binding to ghosts is essentially complete within 2.5 min of mixing which is slightly faster than the rate of hemolysis measured with intact cells. Approximately 4 X 10(4) binding sites per cell, exhibiting a K 0.5 of 0.7 microM exist; this compares with 50% hematocrit of about 0.3 microM for A-III. Binding is absent in ghosts extracted with Nonidet P-40, but is unaffected by pretreatment of ghosts with either trypsin or elastase.

  2. (/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.

  3. Characteristics of binding of human seminal. cap alpha. -inhibin-92 to human pituitary membranes

    SciTech Connect

    Ramasharma, K.; Li, C.H.

    1987-06-01

    The authors investigated the binding of /sup 125/I-labeled ..cap alpha..-inhibin-92 (a 92-residue peptide) to human pituitary membrane preparations. Unlabeled ..cap alpha..-inhibin-92 competed effectively with the labeled peptide for binding to the membranes. Binding was also inhibited by both ..cap alpha..-inhibin-52 and ..cap alpha..-inhibin-31, but less effectively. Scatchard analysis of the ..cap alpha..-inhibin-92 binding data indicated the presence of high-affinity binding sites (1.35 nM/mg of membrane protein) with an apparent K/sub d/ of 0.37 nM. When /sup 125/I-labeled ..cap alpha..-inhibin-92 was covalently crosslinked to the pituitary membrane preparation with disuccinimidyl suberate and the solubilized labeled receptor complex was analyzed by NaDodSO/sub 4//PAGE under either reducing or nonreducing conditions, a single radioactive band at an apparent molecular weight of 90,000 +/- 5000 observed. These data suggest that human pituitary has specific binding sites for ..cap alpha..-inhibins.

  4. 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.

  5. 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.

  6. Covalent binding of single-walled carbon nanotubes to polyamide membranes for antimicrobial surface properties.

    PubMed

    Tiraferri, Alberto; Vecitis, Chad D; Elimelech, Menachem

    2011-08-01

    We propose an innovative approach to impart nanomaterial-specific properties to the surface of thin-film composite membranes. Specifically, biocidal properties were obtained by covalently binding single-walled carbon nanotubes (SWNTs) to the membrane surface. The SWNTs were first modified by purification and ozonolysis to increase their sidewall functionalities, maximize cytotoxic properties, and achieve dispersion in aqueous solution. A tailored reaction protocol was developed to exploit the inherent moieties of hand-cast polyamide membrane surfaces and create covalent amide bonds with the functionalized SWNTs. The reaction is entirely aqueous-based and entails activation of the carboxylate groups of both the membrane and the nanomaterials to maximize reaction with ethylenediamine. The presence of SWNTs was verified after sonication of the membranes, confirming the strength of the bond between the SWNTs and the membrane surface. Characterization of the SWNT-functionalized surfaces demonstrated the attainment of membranes with novel properties that continued to exhibit high performance in water separation processes. The presence of surface-bound antimicrobial SWNTs was confirmed by experiments using E. coli cells that demonstrated an enhanced bacterial cytotoxicity for the SWNT-coated membranes. The SWNT membranes were observed to achieve up to 60% inactivation of bacteria attached to the membrane within 1 h of contact time. Our results suggest the potential of covalently bonded SWNTs to delay the onset of membrane biofouling during operation.

  7. Binding of plasma membrane lipids recruits the yeast integral membrane protein Ist2 to the cortical ER.

    PubMed

    Fischer, Marcel André; Temmerman, Koen; Ercan, Ebru; Nickel, Walter; Seedorf, Matthias

    2009-08-01

    Recruitment of cytosolic proteins to individual membranes is governed by a combination of protein-protein and protein-membrane interactions. Many proteins recognize phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] at the cytosolic surface of the plasma membrane (PM). Here, we show that a protein-lipid interaction can also serve as a dominant signal for the sorting of integral membrane proteins. Interaction with phosphatidly-inositolphosphates (PIPs) at the PM is involved in the targeting of the polytopic yeast protein Ist2 to PM-associated domains of the cortical endoplasmic reticulum (ER). Moreover, binding of PI(4,5)P(2) at the PM functions as a dominant mechanism that targets other integral membrane proteins to PM-associated domains of the cortical ER. This sorting to a subdomain of the ER abolishes proteasomal degradation and trafficking along the classical secretory (sec) pathway. In combination with the localization of IST2 mRNA to the bud tip and other redundant signals in Ist2, binding of PIPs leads to efficient accumulation of Ist2 at domains of the cortical ER from where the protein may reach the PM independently of the function of the sec-pathway.

  8. Binding of vinyl polymers to anionic model membranes.

    PubMed

    Torrens, F; Campos, A; Abad, C

    2003-09-01

    The association of poly2-vinylpyridine (P2VPy) and poly4-vinylpyridine (P4VPy) to dimyristoylphosphatidic acid (DMPA) small unilamellar vesicles (SUVs) was studied as a function of pH, ionic strength (I), polymer concentration and temperature using spectrofluorimetry. Poly(vinylpyridine) (PVPy) data were transformed into association isotherms and analyzed in terms of binding and partition models. In the case of polyions, the inclusion of the activity coefficient in both models was essential. Moreover, a relating equation was proposed to compare parameters based on both theoretical approaches. On the basis of the results obtained, a model was developed to analyze polymer adsorption at the surface level, in which the length of the hydrophobic chain and the position of the N atom in the pyridinium ring play an important role. Transition temperature (Tc) for DMPA (ca. 55 degrees C) is decreased between 15 degrees C-19 degrees C in the presence of PVPy. Van't Hoff isochore showed that the binding constant (KA) accounted for average PVPy-DMPA two-dimensional solid and liquid interactions. KA decreased with I in the presence of both polymers, but was more sensitive to I in the case of P2VPy. Likewise, the number of phospholipid heads (N) involved in the binding process decreased with I in the presence of PVPy. The influence of I was more significant on N than on KA.

  9. Association of Plasmodium berghei proteins with the host erythrocyte membrane: binding to inside-out vesicles.

    PubMed

    Wiser, M F; Sartorelli, A C; Patton, C L

    1990-01-01

    Two acidic phosphoproteins of Plasmodium berghei origin, of 65 and 46 kDa, are associated with the plasma membrane of the host mouse erythrocyte. The 65-kDa protein partitions between a soluble and particulate phase upon host cell lysis, whereas the 46-kDa protein is localized exclusively in the particulate fraction. Both proteins bind to inside-out vesicles derived from erythrocyte ghosts and the conditions of the reassociation reaction indicate that the binding is specific and that the proteins interact only with the cytoplasmic face of the erythrocyte membrane. The 65-kDa protein appears to exist in two membrane-associated states; one loosely bound, which readily dissociates from the membrane, and a more tightly associated state, which does not dissociate under non-denaturing conditions. The 46-kDa protein is tightly bound to the host erythrocyte membrane and does not dissociate. Cross-linking studies suggest that both of these parasite proteins interact with the submembrane cytoskeleton of the erythrocyte, and that the 65-kDa protein also appears to interact simultaneously with the lipid bilayer and erythrocyte membrane proteins. However, direct interaction between the malarial proteins and distinct erythrocyte membrane proteins could not be demonstrated. In summary, these findings indicate that the acidic phosphoproteins of the malarial parasite interact with the cytoplasmic face of the erythrocyte membrane both in vivo and in vitro.

  10. Binding of AP-2 adaptor complex to brain membrane is regulated by phosphorylation of proteins

    SciTech Connect

    Alberdi, A. . E-mail: aalberdi@fcm.uncu.edu.ar; Sartor, T.; Sosa, M.A.

    2005-05-13

    Phosphorylation of proteins appears as a key process in early steps of clathrin coated vesicle formation. Here, we report that treatment of post-nuclear fraction with alkaline phosphatase induced redistribution of {alpha} subunits of AP-2 adaptor complex to cytosol and this effect was higher in the {alpha}2 subunit. A high serine phosphorylation status of {alpha} subunits correlated with the higher affinity of AP-2 to membranes. Using a simple binding assay, where membranes were incubated with either purified adaptors or cytosols, we observed an inhibitory effect of tyrphostin, a tyrosine kinase inhibitor, on the binding of AP-2 to membranes, but also an unexpected decrease induced by the phosphatase inhibitor cyclosporine. We also show an inhibitory effect of ATP mediated by cytosolic proteins, although it could not be related to the phosphorylation of AP-2, suggesting an action upstream a cascade of phosphorylations that participate in the regulation of the assembly of AP-2 to membranes.

  11. Membrane binding sites for plasma lipoproteins on endosomes from rat liver.

    PubMed Central

    Jaeckle, S; Brady, S E; Havel, R J

    1989-01-01

    Highly purified endosomal membranes from rat liver, enriched in receptors for a number of macromolecules taken up into hepatocytes via the coated pit/endosome/lysosome pathway [including the receptor for low density lipoproteins (LDL)], were used to characterize binding sites for lipoproteins containing apolipoprotein E. In endosomal membranes from livers of estradiol-treated rats, in which LDL receptors are induced manyfold, two high-affinity binding sites were found for two apolipoprotein E-rich lipoproteins: very low density beta-lipoproteins (beta-VLDL) from cholesterol-fed rabbits and rat chylomicron remnants. One of these sites, binding to which is inhibited by 30 mM EDTA, appears identical to the LDL receptor by ligand and immunoblotting and other characteristics. The other site, highly resistant to EDTA, does not bind LDL. Binding to the EDTA-resistant site, however, is readily inhibited by heparin (as is the LDL receptor) and also by antisera prepared against rat or bovine LDL receptor. The distribution of the EDTA-resistant site among early endosomes, late endosomes, and endosome-derived receptor-recycling membranes is similar to that of the LDL receptor and other recycling receptors. The LDL receptor was present in endosomal membranes from livers of untreated rats at about 10% of the level found in membranes from estradiol-treated rats, but the EDTA-resistant site was barely detectable. No saturable binding of beta-VLDL that could not be inhibited by antisera to the LDL receptor could be detected in endosomal membranes from livers of either untreated or estradiol-treated rats. The EDTA-resistant site may be a modified form of the LDL receptor that recognizes apolipoprotein E but not the B apolipoprotein of LDL. Alternatively, it may be a distinct receptor sharing immunological determinants with the LDL receptor, specialized for the endocytosis of certain lipoproteins containing apolipoprotein E, including chylomicron remnants. Images PMID:2538819

  12. 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.

  13. Distribution of adenylate cyclase and GTP-binding proteins in hepatic plasma membranes.

    PubMed

    Dixon, B S; Sutherland, E; Alexander, A; Nibel, D; Simon, F R

    1993-10-01

    Hepatic membrane subfractions prepared from control rats demonstrated forskolin (FSK)-stimulated adenylate cyclase activity in the basolateral (sinusoidal) but not apical (canalicular) plasma membrane. After bile duct ligation (BDL) for 12 or 24 h, there was an increase in FSK-stimulated adenylate cyclase activity in the apical membrane (54.2 +/- 3.9 pmol.mg-1 x min-1). The mechanism for this increase was explored further. ATP hydrolysis was found to be much higher in the apical than the basolateral membrane. Increasing the ATP levels in the assay enhanced apical membrane adenylate cyclase activity (10.5 +/- 0.2 pmol.mg-l.min-1); however, total adenosinetriphosphatase (ATPase) activity was not altered after BDL. Extraction of the apical membrane with bile acids or other detergents resulted in a two- to threefold increase in adenylate cyclase activity (30.6 +/- 3.6 pmol.mg-1 x min-1; detergent C12E8) This suggested that bile duct ligation was acting via the detergent-like action of bile acids to uncover latent adenylate cyclase activity on apical membranes. Further studies demonstrated that both BDL and detergent extraction also enhanced toxin-directed ADP-ribosylation of Gs alpha (cholera toxin) and Gi alpha (pertussis toxin) in the apical but not the basolateral membrane. After BDL, Gi alpha was found to be twofold greater in the apical membrane than the basolateral membrane. Immunoblotting using specific G protein antibodies further confirmed that apical membranes from control rats had a higher concentration of Gi1, 2 alpha and beta and slightly elevated levels of Gi3 alpha and Gs alpha compared with the basolateral membrane. The results demonstrate that adenylate cyclase and heterotrimeric GTP-binding proteins are present on the apical membrane, but measurement of their functional activity requires detergent permeabilization of apical membrane vesicles and is limited by the presence of high ATPase activity.

  14. Glucocorticoid interactions with ethanol effects on synaptic plasma membranes: influence on [125I]calmodulin binding.

    PubMed

    Sze, P Y

    1996-02-01

    Ca(++)-dependent binding of calmodulin (CaM) to brain synaptic plasma membranes is known to be inhibited by ethanol and stimulated by glucocorticoids. These opposite neurochemical actions between ethanol and the steroids in vitro are consistent with glucocorticoid antagonism of ethanol-induced sedation reported to occur in vivo. The present study was undertaken to characterize the interactions of corticosterone with ethanol effects on [125I]CaM binding in synaptic plasma membranes. From the shift of concentration-response curves when corticosterone and ethanol were present in combination, the interaction between steroid stimulation and ethanol inhibition occurred in an additive relationship over the range of their effective concentrations. From Scatchard analyses, ethanol-induced decrease in membrane affinity for [125I]CaM was antagonized by steroid-induced increase in the membrane affinity, indicating that the convergent event in their interaction was the alteration of membrane affinity for CaM. Glucocorticoid antagonism of ethanol inhibition of [125I]CaM binding exhibited a high degree of steroid specificity; steroids with glucocorticoid activity including cortisol, dexamethasone and triamcinolone were effective, whereas gonadal steroids and excitatory neuroactive steroid metabolites were ineffective. The demonstration that glucocorticoids antagonized the inhibition of CaM binding by ethanol provides support for the hypothesis that these steroids are among the endogenous factors that modulate neuronal sensitivity to ethanol.

  15. Binding of thyroglobulin to bovine thyroid membranes. Role of specific amino acids in receptor recognition.

    PubMed

    Shifrin, S; Kohn, L D

    1981-10-25

    Bovine thyroglobulin was treated with increasing ratios of succinic anhydride, trinitrobenzene sulfonic acid, tetranitromethane, and N-acetylimidazole in an attempt to assess the role of lysine or tyrosine residues in binding to thyroid membrane receptors. Extensive succinylation results in dissociation to 12 S thyroglobulin with retention of a considerable portion of the three-dimensional structure. Only 25% of the lysine residues can be modified by trinitrophenylation without affecting inter-subunit interactions. Succinylation as well as trinitrophenylation increases the affinity of thyroglobulin for the membrane receptor by a factor of 2. The binding of thyroglobulin to the membrane was reduced after nitration of 30% of the tyrosyl residues with tetranitromethane. O-Acetylation of 40-70% of the tyrosyl residues by N-acetylimidazole nearly abolished the ability of thyroglobulin to bind to the membrane. Removal of the O-acetyl group with hydroxylamine restored the binding properties. The results indicate that tyrosyl residues play an important role in thyroglobulin interactions with thyroid membranes.

  16. Conformational antibody binding to a native, cell-free expressed GPCR in block copolymer membranes.

    PubMed

    de Hoog, Hans-Peter M; Lin JieRong, Esther M; Banerjee, Sourabh; Décaillot, Fabien M; Nallani, Madhavan

    2014-01-01

    G-protein coupled receptors (GPCRs) play a key role in physiological processes and are attractive drug targets. Their biophysical characterization is, however, highly challenging because of their innate instability outside a stabilizing membrane and the difficulty of finding a suitable expression system. We here show the cell-free expression of a GPCR, CXCR4, and its direct embedding in diblock copolymer membranes. The polymer-stabilized CXCR4 is readily immobilized onto biosensor chips for label-free binding analysis. Kinetic characterization using a conformationally sensitive antibody shows the receptor to exist in the correctly folded conformation, showing binding behaviour that is commensurate with heterologously expressed CXCR4.

  17. Binding of Escherichia coli heat-stable enterotoxin to rat intestinal cells and brush border membranes.

    PubMed Central

    Frantz, J C; Jaso-Friedman, L; Robertson, D C

    1984-01-01

    The association of heat-stable enterotoxin (STa) produced by enterotoxigenic Escherichia coli 431 with isolated rat intestinal epithelial cells and brush border membranes was characterized. Specific binding of strain 431 125I-STa to a single class of specific high-affinity receptors was saturable and temperature dependent and reached a maximum between 5 and 10 min. A 1,000-fold excess of unlabeled 431 STa competitively displaced 90 to 95% of radiolabeled enterotoxin bound to brush border membranes. In contrast, specific binding of 431 125I-STa to intestinal cells ranged from 40 to 65%. The number of STa-specific receptors on rat intestinal cells determined by Scatchard analysis was 47,520 +/- 14,352 (mean +/- standard error of the mean) per cell, with affinity constants (KaS) of 2.55 X 10(11)and 4.32 x 10(11) liters/mol determined for intestinal cells and brush border membranes, respectively. Villus intestinal cells appeared to possess about twice as many STa receptors as did crypt cells. Dissociation of specifically bound 431 125I-STa from intestinal cells and brush border membranes was minimal (2 to 5%). In addition, neither the rate nor the extent of dissociation was increased by a 1,000-fold excess of unlabeled homologous 431 Sta. Binding experiments with 431 125I-STa and brush border membranes showed that purified unlabeled STas from enterotoxigenic E. coli strains 667 (class 1 porcine enteropathogen), B-41 (bovine enteropathogen), and human strains 213C2 (Mexico) and 153961-2 (Dacca, Bangledesh) exhibited patterns of competitive inhibition similar to those of homologous unlabeled 431 STa (class 2 enteropathogen). A lipid extract which contained gangliosides and glycolipids exhibited dose-dependent competitive inhibition of heat-labile enterotoxin binding to brush border membranes but did not inhibit binding of 431 125I-STa. Purified heat-labile enterotoxin from strain 286C2 did not inhibit binding of 431 STa to brush border membranes. Pronase treatment of

  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. Molecular biology of androgen insensitivity.

    PubMed

    Jääskeläinen, Jarmo

    2012-04-16

    Androgen insensitivity syndrome (AIS) is the most common specific cause of 46,XY disorder in sex development. The androgen signaling pathway is complex but so far, the only gene linked with AIS is the androgen receptor (AR). Mutations in the AR are found in most subjects with complete AIS but in partial AIS, the rate has varied 28-73%, depending on the case selection. More than 400 different mutations in AR leading to AIS have been reported. Most mutations are missense substitutions located in the ligand binding domain of the receptor. However, when systematically screened, a substantial amount of mutations can be detected also in the N-terminal domain encoded by exon 1. Within this exon lie two trinucleotide, CAG and GGN repeat regions which are polymorphic in length. Their role in androgen insensitivity is somewhat unclear. Recent advances in protein modeling have resulted in better understanding of the mechanism of known AR mutations.

  20. ADP-ribosylation factor, a small GTP-binding protein, is required for binding of the coatomer protein beta-COP to Golgi membranes.

    PubMed Central

    Donaldson, J G; Cassel, D; Kahn, R A; Klausner, R D

    1992-01-01

    The coatomer is a cytosolic protein complex that reversibly associates with Golgi membranes and is implicated in modulating Golgi membrane transport. The association of beta-COP, a component of coatomer, with Golgi membranes is enhanced by guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), a nonhydrolyzable analogue of GTP, and by a mixture of aluminum and fluoride ions (Al/F). Here we show that the ADP-ribosylation factor (ARF) is required for the binding of beta-COP. Thus, beta-COP contained in a coatomer fraction that has been resolved from ARF does not bind to Golgi membranes, whereas binding can be reconstituted by the addition of recombinant ARF. Furthermore, an N-terminal peptide of ARF, which blocks ARF binding to Golgi membranes, inhibits GTP[gamma S]- as well as the Al/F-enhanced binding of beta-COP. We show that Golgi coat protein binding involves a sequential reaction where an initial interaction of ARF and GTP[gamma S] with the membrane allows subsequent binding of beta-COP to take place in the absence of free ARF and GTP[gamma S]. The fungal metabolite brefeldin A, which is known to prevent the association of coat proteins with Golgi membrane, is shown to exert this effect by interfering with the initial ARF-membrane interaction step. Images PMID:1631136

  1. 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

  2. Tyrosine Hydroxylase Binding to Phospholipid Membranes Prompts Its Amyloid Aggregation and Compromises Bilayer Integrity

    PubMed Central

    Baumann, Anne; Jorge-Finnigan, Ana; Jung-KC, Kunwar; Sauter, Alexander; Horvath, Istvan; Morozova-Roche, Ludmilla A.; Martinez, Aurora

    2016-01-01

    Tyrosine hydroxylase (TH), a rate-limiting enzyme in the synthesis of catecholamine neurotransmitters and hormones, binds to negatively charged phospholipid membranes. Binding to both large and giant unilamellar vesicles causes membrane permeabilization, as observed by efflux and influx of fluorescence dyes. Whereas the initial protein-membrane interaction involves the N-terminal tail that constitutes an extension of the regulatory ACT-domain, prolonged membrane binding induces misfolding and self-oligomerization of TH over time as shown by circular dichroism and Thioflavin T fluorescence. The gradual amyloid-like aggregation likely occurs through cross-β interactions involving aggregation-prone motives in the catalytic domains, consistent with the formation of chain and ring-like protofilaments observed by atomic force microscopy in monolayer-bound TH. PC12 cells treated with the neurotoxin 6-hydroxydopamine displayed increased TH levels in the mitochondrial fraction, while incubation of isolated mitochondria with TH led to a decrease in the mitochondrial membrane potential. Furthermore, cell-substrate impedance and viability assays showed that supplementing the culture media with TH compromises cell viability over time. Our results revealed that the disruptive effect of TH on cell membranes may be a cytotoxic and pathogenic factor if the regulation and intracellular stability of TH is compromised. PMID:28004763

  3. Lipid binding and membrane penetration of polymyxin B derivatives studied in a biomimetic vesicle system.

    PubMed Central

    Katz, Marina; Tsubery, Haim; Kolusheva, Sofiya; Shames, Alex; Fridkin, Mati; Jelinek, Raz

    2003-01-01

    Understanding membrane interactions and cell-wall permeation of Gram-negative bacteria is of great importance, owing to increasing bacterial resistance to existing drugs and therapeutic treatments. Here we use biomimetic lipid vesicles to analyse membrane association and penetration by synthetic derivatives of polymyxin B (PMB), a potent naturally occurring antibacterial cyclic peptide. The PMB analogues studied were PMB nonapeptide (PMBN), in which the hydrophobic alkyl residue was cleaved, PMBN diastereomer containing D-instead of L-amino acids within the cyclic ring (dPMBN) and PMBN where the hydrophobic alkyl chain was replaced with an Ala6 repeat (Ala6-PMBN). Peptide binding measurements, colorimetric transitions induced within the vesicles, fluorescence quenching experiments and ESR spectroscopy were applied to investigate the structural parameters underlying the different membrane-permeation profiles and biological activities of the analogues. The experiments point to the role of negatively charged lipids in membrane binding and confirm the prominence of lipopolisaccharide (LPS) in promoting membrane association and penetration by the peptides. Examination of the lipid interactions of the PMB derivatives shows that the cyclic moiety of PMB is not only implicated in lipid attachment and LPS binding, but also affects penetration into the inner bilayer core. The addition of the Ala6 peptide moiety, however, does not significantly promote peptide insertion into the hydrophobic lipid environment. The data also indicate that the extent of penetration into the lipid bilayer is not related to the overall affinity of the peptides to the membrane. PMID:12848621

  4. Drug binding and mobility relating to the thermal fluctuation in fluid lipid membranes

    NASA Astrophysics Data System (ADS)

    Okamura, Emiko; Yoshii, Noriyuki

    2008-12-01

    Drug binding and mobility in fluid lipid bilayer membranes are quantified in situ by using the multinuclear solution NMR combined with the pulsed-field-gradient technique. One-dimensional and pulsed-field-gradient F19 and H1 NMR signals of an anticancer drug, 5-fluorouracil (5FU) are analyzed at 283-313 K in the presence of large unilamellar vesicles (LUVs) of egg phosphatidylcholine (EPC) as model cell membranes. The simultaneous observation of the membrane-bound and free 5FU signals enables to quantify in what amount of 5FU is bound to the membrane and how fast 5FU is moving within the membrane in relation to the thermal fluctuation of the soft, fluid environment. It is shown that the mobility of membrane-bound 5FU is slowed down by almost two orders of magnitude and similar to the lipid movement in the membrane, the movement closely related to the intramembrane fluidity. The mobility of 5FU and EPC is, however, not similar at 313 K; the 5FU movement is enhanced in the membrane as a result of the loose binding of 5FU in the lipid matrices. The membrane-bound fraction of 5FU is ˜0.1 and almost unaltered over the temperature range examined. It is also independent of the 5FU concentration from 2 to 30 mM with respect to the 40-50 mM LUV. The free energy of the 5FU binding is estimated at -4 to -2 kJ/mol, the magnitude always close to the thermal fluctuation, 2.4-2.6 kJ/mol.

  5. 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.

  6. The role of androgen and androgen receptor in skin-related disorders.

    PubMed

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

    2012-09-01

    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.

  7. Num1 anchors mitochondria to the plasma membrane via two domains with different lipid binding specificities.

    PubMed

    Ping, Holly A; Kraft, Lauren M; Chen, WeiTing; Nilles, Amy E; Lackner, Laura L

    2016-06-06

    The mitochondria-ER cortex anchor (MECA) is required for proper mitochondrial distribution and functions by tethering mitochondria to the plasma membrane. The core component of MECA is the multidomain protein Num1, which assembles into clusters at the cell cortex. We show Num1 adopts an extended, polarized conformation. Its N-terminal coiled-coil domain (Num1CC) is proximal to mitochondria, and the C-terminal pleckstrin homology domain is associated with the plasma membrane. We find that Num1CC interacts directly with phospholipid membranes and displays a strong preference for the mitochondria-specific phospholipid cardiolipin. This direct membrane interaction is critical for MECA function. Thus, mitochondrial anchoring is mediated by a protein that interacts directly with two different membranes through lipid-specific binding domains, suggesting a general mechanism for interorganelle tethering.

  8. Num1 anchors mitochondria to the plasma membrane via two domains with different lipid binding specificities

    PubMed Central

    Ping, Holly A.; Kraft, Lauren M.; Chen, WeiTing; Nilles, Amy E.

    2016-01-01

    The mitochondria–ER cortex anchor (MECA) is required for proper mitochondrial distribution and functions by tethering mitochondria to the plasma membrane. The core component of MECA is the multidomain protein Num1, which assembles into clusters at the cell cortex. We show Num1 adopts an extended, polarized conformation. Its N-terminal coiled-coil domain (Num1CC) is proximal to mitochondria, and the C-terminal pleckstrin homology domain is associated with the plasma membrane. We find that Num1CC interacts directly with phospholipid membranes and displays a strong preference for the mitochondria-specific phospholipid cardiolipin. This direct membrane interaction is critical for MECA function. Thus, mitochondrial anchoring is mediated by a protein that interacts directly with two different membranes through lipid-specific binding domains, suggesting a general mechanism for interorganelle tethering. PMID:27241910

  9. 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

  10. 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

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

    SciTech Connect

    Dick, Robert A.; Datta, Siddhartha A. K.; Nanda, Hirsh; Fang, Xianyang; Wen, Yi; Barros, Marilia; Wang, Yun-Xing; Rein, Alan; Vogt, Volker M.; Sundquist, W. I.

    2016-05-06

    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 interactionsin vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particlesin 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 interactionin vitro, either by directly contacting acidic lipids or by promoting Gag multimerization.

    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

  12. Does Fluoride Affect Serum Testosterone and Androgen Binding Protein with Age-Specificity? A Population-Based Cross-Sectional Study in Chinese Male Farmers.

    PubMed

    Duan, Leizhen; Zhu, Jingyuan; Wang, Keyan; Zhou, Guoyu; Yang, Yuejin; Cui, Liuxin; Huang, Hui; Cheng, Xuemin; Ba, Yue

    2016-12-01

    Many studies have demonstrated that exposure to excess fluoride was associated with a variety of diseases. Little is known about the variation of testosterone (T) levels caused by fluoride exposure. The aim of this study is to explore the association of fluoride exposure and age with serum T and androgen-binding protein (ABP) levels in male farmers. A cross-sectional study was conducted in a county of Henan Province, China, including high fluoride exposure from drinking water villages and control villages. Male farmers aged 18-55 years old who lived in these villages were recruited by cluster sampling and divided into a higher fluoride exposure group (HFG) and a lower fluoride exposure group (LFG) according to the level of urinary fluoride. Levels of T and ABP in serum were measured using chemiluminescence immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) respectively. Markedly lower T levels were observed in male farmers from the HFG than in those from the LFG (t = 2.496, P < 0.05). Furthermore, younger farmers, 18-29 and 30-39 years old, may be the most likely to have lower T levels when exposed to fluoride (P < 0.05). No significant differences were observed in serum ABP levels in all male farmers between the two groups with different fluoride exposure. These results supported that excess fluoride exposure decreased serum T levels of male farmers with age-specificity.

  13. Evolution of the ABPA subunit of androgen-binding protein expressed in the submaxillary glands in New and Old World rodent taxa.

    PubMed

    Vandewege, Michael W; Phillips, Carleton J; Wickliffe, Jeffrey K; Hoffmann, Federico G

    2013-05-01

    The salivary androgen-binding proteins (ABPs) are members of the secretoglobin gene family present in mammals. Each ABP is a heterodimer assembled as an ABPA subunit encoded by an Abpa gene and linked by disulfide bridges to an ABPBG subunit encoded by an Abpbg gene. The ABP dimers are secreted into the saliva of mice and then transferred to the pelage after grooming and subsequently to the environment allowing an animal to mark territory with a biochemical signal. The putative role of the mouse salivary ABPs is that of pheromones mediating mate selection resulting in assortative mating in the Mus musculus species complex. We focused on comparing patterns of molecular evolution between the Abpa genes expressed in the submaxillary glands of species of New World and Old World muroids. We found that in both sets of rodents the Abpa genes expressed in the submaxillary glands appear to be evolving under a similar evolutionary regime, with relatively high nonsynonymous substitution rates, suggesting that ABP might play a similar biological role in both systems. Thus, ABP could be involved with mate recognition and species isolation in New World as well as Old World muroids.

  14. Sialic acid mediates the initial binding of positively charged inorganic particles to alveolar macrophage membranes.

    PubMed

    Gallagher, J E; George, G; Brody, A R

    1987-06-01

    Pulmonary macrophages phagocytize inhaled particles and are postulated to play a role in the development of pulmonary interstitial fibrogenesis. The basic biologic mechanisms through which inhaled particles bind to macrophage membranes and subsequently are phagocytized remain unclear. We hypothesize that positively charged particles bind to negatively charged sialic acid (SA) residues on macrophage membranes. Alveolar Macrophages (AM) were collected by saline lavage from normal rat lungs. The cells adhered to plastic coverslips in serum-free phosphate buffered saline at 37 degrees C for 45 min and then were maintained at 4 degrees C for the binding experiments. Even distribution of SA groups on AM surfaces was demonstrated by scanning electron microscopy of wheat germ agglutinin (WGA) conjugated to 50 nm gold spheres. The WGA is a lectin that binds specifically to sialic acid, and pretreatment of AM with this lectin prevented the binding of positively charged carbonyl iron (C-Fe) spheres, aluminum (Al) spheres, and chrysotile asbestos fibers to AM surfaces. Limulus protein, another lectin with binding specificity for SA, similarly blocked the binding of positively charged spheres and chrysotile asbestos fibers but not negatively charged glass spheres or crocidolite asbestos fibers. Con A and ricin, lectins that bind to mannose and galactose residues, respectively, did not block particle binding. When both positively charged iron spheres and negatively charged glass spheres were prebound to AM membranes, subsequent treatment with WGA displaced only the positively charged spheres from macrophage surfaces. Con A and ricin had no effect on prebound positively charged C-Fe and Al spheres.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Two-Step Membrane Binding of NDPK-B Induces Membrane Fluidity Decrease and Changes in Lipid Lateral Organization and Protein Cluster Formation.

    PubMed

    Francois-Moutal, Liberty; Ouberai, Myriam M; Maniti, Ofelia; Welland, Mark E; Strzelecka-Kiliszek, Agnieszka; Wos, Marcin; Pikula, Slawomir; Bandorowicz-Pikula, Joanna; Marcillat, Olivier; Granjon, Thierry

    2016-12-06

    Nucleoside diphosphate kinases (NDPKs) are crucial elements in a wide array of cellular physiological or pathophysiological processes such as apoptosis, proliferation, or metastasis formation. Among the NDPK isoenzymes, NDPK-B, a cytoplasmic protein, was reported to be associated with several biological membranes such as plasma or endoplasmic reticulum membranes. Using several membrane models (liposomes, lipid monolayers, and supported lipid bilayers) associated with biophysical approaches, we show that lipid membrane binding occurs in a two-step process: first, initiation by a strong electrostatic adsorption process and followed by shallow penetration of the protein within the membrane. The NDPK-B binding leads to a decrease in membrane fluidity and formation of protein patches. The ability of NDPK-B to form microdomains at the membrane level may be related to protein-protein interactions triggered by its association with anionic phospholipids. Such accumulation of NDPK-B would amplify its effects in functional platform formation and protein recruitment at the membrane.

  16. 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

  17. G-protein alpha-s and -12 subunits are involved in androgen-stimulated PI3K activation and androgen receptor transactivation in prostate cancer cells

    PubMed Central

    Liu, Jianjun; Youn, Hyewon; Yang, Jun; Du, Ningchao; Liu, Jihong; Liu, Hongwei; Li, Benyi

    2011-01-01

    BACKGROUND The androgen receptor (AR) is a ligand-dependent transcription factor that mediates androgenic hormone action in cells. We recently demonstrated the involvement of phosphoinositide 3-OH kinase (PI3K) p110beta in AR transactivation and gene expression. In this study, we determined the upstream signals that lead to PI3K/p110beta activation and AR transactivation after androgen stimulation. METHODS Human prostate cancer LAPC-4 and 22Rv1 cell lines were used for the experiments. AR transactivation was assessed using an androgen responsive element-driven luciferase (ARE-LUC) assay. Cell proliferation was examined using BrdU incorporation and MTT assays. Target genes were silenced using small interfering RNA (siRNA) approach. Gene expression was evaluated at the mRNA level (real-time RT-PCR) and protein level (Western blot). PI3K kinase activities were measured using immunoprecipitation-based in vitro kinase assay. The AR-DNA binding activity was determined using Chromatin-immunoprecipitation (ChIP) assay. RESULTS First, at the cellular plasma membrane, disrupting the integrity of caveolae microdomain with methyl-β- cyclodextrin (M-β-CD) abolished androgen-induced AR transactivation and gene expression. Then, knocking down caveolae structural proteins caveolin-1 or -2 with the gene-specific siRNAs significantly reduced androgen-induced AR transactivation. Next, silencing Gαs and Gα12 genes but not other G-proteins blocked androgen-induced AR transactivation and cell proliferation. Consistently, overexpression of Gαs or Gα12 active mutants enhanced androgen-induced AR transactivation, of which Gαs active mutant sensitized the AR to castration-level of androgen (R1881). Most interestingly, knocking down Gαs but not Gα12 subunit significantly suppressed androgen-stimulated PI3K p110beta activation. However, chromatin-immunoprecipitation (ChIP) analysis revealed that both Gαs or Gα12 subunits are involved in androgen-induced AR interaction with the AR

  18. 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.

  19. Monitoring membrane binding and insertion of peptides by two-color fluorescent label.

    PubMed

    Postupalenko, V Y; Shvadchak, V V; Duportail, G; Pivovarenko, V G; Klymchenko, A S; Mély, Y

    2011-01-01

    Herein, we developed an approach for monitoring membrane binding and insertion of peptides using a fluorescent environment-sensitive label of the 3-hydroxyflavone family. For this purpose, we labeled the N-terminus of three synthetic peptides, melittin, magainin 2 and poly-l-lysine capable to interact with lipid membranes. Binding of these peptides to lipid vesicles induced a strong fluorescence increase, which enabled to quantify the peptide-membrane interaction. Moreover, the dual emission of the label in these peptides correlated well with the depth of its insertion measured by the parallax quenching method. Thus, in melittin and magainin 2, which show deep insertion of their N-terminus, the label presented a dual emission corresponding to a low polar environment, while the environment of the poly-l-lysine N-terminus was rather polar, consistent with its location close to the bilayer surface. Using spectral deconvolution to distinguish the non-hydrated label species from the hydrated ones and two photon fluorescence microscopy to determine the probe orientation in giant vesicles, we found that the non-hydrated species were vertically oriented in the bilayer and constituted the best indicators for evaluating the depth of the peptide N-terminus in membranes. Thus, this label constitutes an interesting new tool for monitoring membrane binding and insertion of peptides.

  20. Isolation of an actin-binding protein from membranes of Dictyostelium discoideum

    PubMed Central

    1985-01-01

    We prepared a probe of radiolabeled, glutaraldehyde cross-linked filamentous actin (F-actin) to study binding of actin to membranes of Dictyostelium discoideum. The probe bound to membranes or detergent extracts of membranes with a high affinity and in a saturable manner. The binding could be reduced by boiling of either the actin probe or the membranes, or by addition of excess native F-actin, but not by addition of an equivalent amount of bovine serum albumin, to the assay. The probe labeled several proteins when used to overlay sodium dodecyl sulfate gels of Dictyostelium membranes. One of these labeled proteins was a 24,000-mol-wt protein (p24), which was soluble only in the presence of a high concentration of sodium deoxycholate (5%, wt/vol) at room temperature or above. The p24 was purified by selective detergent extraction and column chromatography. When tested in a novel two-phase binding assay, p24 bound both native monomeric actin (G-actin) and F- actin in a specific manner. In this assay, G-actin bound p24 with a submicromolar affinity. PMID:3972891

  1. Investigations of the binding of 239Pu to liver cell membranes.

    PubMed

    Planas-Bohne, F; Kampmann, G; Olinger, H

    1989-07-15

    The binding of Pu to liver cell membranes was studied and compared with that of iron with which plutonium shares some physiological properties. The binding of both metals is sensitive to pH changes and they can be dissociated from their binding sites by chelating agents and transferrin. The metal-binding proteins can be extracted with detergents. Both metals have at least two binding sites, the molecular weights of which lie between 150 and 400 kDa; the isoelectric points for iron are 5.5 and 6.5, and for plutonium 6.0 and 6.5. The significance of these results for plutonium uptake into liver cells is discussed.

  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.

  4. 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.

  5. A human endothelial cell membrane protein that binds Staphylococcus aureus in vitro.

    PubMed Central

    Tompkins, D C; Hatcher, V B; Patel, D; Orr, G A; Higgins, L L; Lowy, F D

    1990-01-01

    We have investigated S. aureus adherence to human endothelial cells utilizing an in vitro model. Staphylococcus binding to confluent endothelial cell monolayers was saturable in both dose and time response studies suggesting that the binding interaction was specific. We have developed a technique, based on the pH dependent affinity of iminobiotin for streptavidin, for the isolation of an endothelial cell membrane component that binds S. aureus, in vitro. A 50-kD membrane component was isolated and purified using this approach. This component was trypsin sensitive, periodate insensitive, and did not label with [3H]glucosamine. [35S]Methionine and [125I]iodine labeling confirmed that the protein was synthesized by and expressed on the endothelial cell surface. Functional binding studies demonstrated that staphylococci, but not endothelial cells, bound to the protein when immobilized on microtiter wells. Preincubation of staphylococci with the purified protein significantly (P less than 0.001) reduced staphylococcal binding to cultured endothelial cells. The capacity of S. aureus to colonize and invade endovascular surfaces may in part be a consequence of staphylococcal interaction with this endothelial cell membrane protein. Images PMID:2318978

  6. High affinity binding of 125I-angiotensin II to rat glomerular basement membranes.

    PubMed Central

    Sraer, J; Baud, L; Cosyns, J P; Verroust, P; Nivez, M P; Ardaillou, R

    1977-01-01

    125I-angiotensin II (AII) specifically bound to rat glomerular basement membrane (GBM). The kinetics of binding were similar to those obtained with the total glomeruli. The apparent dissociation constant was close to 50 pM with both preparations. The number of sites related to the amount of protein was two times greater with GBM than with total glomeruli. Since the amount of GBM protein extracted from a given amount of glomerular protein was about 10%, it was possible to estimate the share of the GBM binding sites for AII as representing 20% of the total number present in the entire glomerulus. Binding studies at equilibrium as a function of 125I-AII concentration and competitive binding experiments suggested either multiplicity of the binding sites or cooperativity in the binding reaction. Degradation of 125I-AII in the presence of GBM was slight and did not increase with time. The difference in the degrees of degradation of 125I-AII was too small to account for the observed difference in binding when the results obtained with GBM and isolated glomeruli preparations were compared. 125I-AII binding to GBM was increased after treatment of these membranes with collagenase, slightly diminished with neuraminidase, and almost completely abolished with trypsin suggesting the proteic nature of the receptor. 125I-AII binding to GBM was diminished after incubation of GBM with anti-GBM antibodies as a result of a decrease in the number of binding sites. 125I-AII binding was even more diminished in preparations of glomeruli isolated from rats passively immunized with anti-GBM antibodies when compared with glomeruli from control animals. This resulted from both smaller affinity for AII and decrease in the number of the binding sites. The present data provides evidence for specific binding sites for AII localized on GBM. This is noteworthy since receptors for polypeptide hormones are currently observed on the surface of cell membranes. These findings also suggest a new

  7. Binding of (/sup 3/H)Forskolin to rat brain membranes

    SciTech Connect

    Seamon, K.B.; Vaillancourt, R.; Edwards, M.; Daly, J.W.

    1984-08-01

    (12-/sup 3/H)Forskolin (27 Ci/mmol) has been used to study binding sites in rat brain tissue by using both centrifugation and filtration assays. The binding isotherm measured in the presence of 5 mM MgCl/sub 2/ by using the centrifugation assay is described best by a two-site model: K/sub d1/ = 15 nM, B/sub max/sub 1// (maximal binding) = 270 fmol/mg of protein; K/sub d2/ = 1.1 ..mu..M; B/sub max/sub 2// = 4.2 pmol/mg of protein. Only the high-affinity binding sites are detected when the binding is determined by using a filtration assay; K/sub d/ = 26 nM, B/sub max/ = 400 fmol/mg of protein. Analogs of forskolin that do not activate adenylate cyclase (EC 4.6.1.1) do not compete effectively for (/sup 3/H)forskolin binding sites. Analogs of forskolin that are less potent than forskolin in activating adenylate cyclase are also less potent in competing for forskolin binding sites. The presence of 5 mM MgCl/sub 2/ or MnCl/sub 2/ was found to enhance binding. In the presence of 1 mM EDTA the amount of high-affinity binding is reduced to 110 fmol/mg of protein with no change in K/sub d/. There is no effect of CaCl/sub 2/ (20 mM) or NaCl (100 mM) on the binding. No high-affinity binding can be detected in membranes from ram sperm, which contains an adenylate cyclase that is not activated by forskolin. It is proposed that the high-affinity binding sites for forskolin are associated with the activated complex of catalytic subunit and stimulatory guanine nucleotide binding protein. 23 references, 5 figures, 2 tables.

  8. Binding of Bacillus thuringiensis Cry1 Toxins to the Midgut Brush Border Membrane Vesicles of Chilo suppressalis (Lepidoptera: Pyralidae): Evidence of Shared Binding Sites

    PubMed Central

    Fiuza, L.; Nielsen-Leroux, C.; Goze, E.; Frutos, R.; Charles, J.

    1996-01-01

    Binding and competition among Cry1Aa, Cry1Ac, and Cry1Ba toxins were analyzed quantitatively in vitro by using (sup125)I-labeled activated toxins and brush border membrane vesicles isolated from Chilo suppressalis larval midguts. The three toxins bound specifically to the midgut brush border membrane vesicles. Direct binding experiments showed that Cry1Aa and Cry1Ba recognized a single class of binding sites with different affinities, whereas Cry1Aa recognized two classes of binding sites, one with a high affinity and a low concentration and the other with a lower affinity but higher concentration. Competition experiments showed that toxins Cry1Ac and Cry1Ba shared a binding site in the C. suppressalis midgut membranes and that this site was also the low-affinity binding site for Cry1Aa. PMID:16535306

  9. 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.

  10. Binding of (/sup 3/H)forskolin to platelet membranes and solubilized proteins from bovine brain

    SciTech Connect

    Nelson, C.A.; Seamon, K.B.

    1986-05-01

    (/sup 3/H)Forskolin ((/sup 3/H)FSK) bound to platelet membranes with a Kd of 20 nM and a Bmax of 125 fmol/mg protein. The Bmax was increased to 400 fmol/mg protein in the presence of GppNHp (or NaF) and MgCl/sub 2/ with no change in Kd. PGE/sub 1/ decreased the EC50 of GppNHp to increase the Bmax for (/sup 3/H)FSK binding from 600 nM to 35 nM. In contrast, PGE/sub 1/ had no effect on the EC50 of NaF to increase (/sup 3/H)FSK binding. (/sup 3/H)FSK binding increased slowly over 60 min when forskolin and GppNHp were added to membranes simultaneously at 20/sup 0/C. Preincubation of membranes with GppNHp at 20/sup 5/C also caused a linear increase in adenylate cyclase specific activity over 60 minutes. (/sup 3/H)FSK bound to solubilized protein from bovine brain membrane with a Kd of 22 nM. GppNHp increased the number of binding sites in solubilized proteins only if membranes were not preincubated with GppNHp prior to solubilization. In conclusion the number of binding sites for (/sup 3/H)FSK is increased by agents that activate adenylate cyclase through the Ns protein. These sites appear to be associated with an activated complex of the Ns protein and adenylate cyclase.

  11. 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

  12. Analysis of Arf1 GTPase-dependent membrane binding and remodeling using the exomer secretory vesicle cargo adaptor

    PubMed Central

    Paczkowski, Jon E.; Fromme, J. Christopher

    2016-01-01

    Summary Protein-protein and protein-membrane interactions play a critical role in shaping biological membranes through direct physical contact with the membrane surface. This is particularly evident in many steps of membrane trafficking, in which proteins deform the membrane and induce fission to form transport carriers. The small GTPase Arf1 and related proteins have the ability to remodel membranes by insertion of an amphipathic helix into the membrane. Arf1 and the exomer cargo adaptor coordinate cargo sorting into subset of secretory vesicle carriers in the model organism Saccharomyces cerevisiae. Here, we detail the assays we used to explore the cooperative action of Arf1 and exomer to bind and remodel membranes. We expect these methods are broadly applicable to other small GTPase/effector systems where investigation of membrane binding and remodeling is of interest. PMID:27632000

  13. Characterization of GTP-binding proteins in Golgi-associated membrane vesicles from rat adipocytes.

    PubMed Central

    Schürmann, A; Rosenthal, W; Schultz, G; Joost, H G

    1992-01-01

    We have previously reported that guanine nucleotides inhibit glucose transport activity reconstituted from adipocyte membrane fractions. In order to further investigate the hypothetical involvement of guanine-nucleotide-binding proteins (GTP-binding proteins) in the regulation of insulin-sensitive glucose transport activity, we studied their subcellular distribution in adipocytes treated or not with insulin. Adipocytes were homogenized and fractionated to yield plasma membranes (PM) and a Golgi-enriched fraction of intracellular membranes (low-density microsomes, LDM). In these membrane fractions, total guanosine 5'-[gamma-[35S]thio]triphosphate ([35S]GTP[S]) binding, alpha- and beta-subunits of heterotrimeric G-proteins, proto-oncogenes Ha-ras and K-ras, and 23-28 kDa GTP-binding proteins were assayed. The levels of alpha s and alpha i (the alpha-subunits of Gs and Gi) were approx. 8-fold lower in LDM than in PM; beta-subunits, Ha-ras and K-ras were not detectable in LDM. Total GTP[S]-binding sites and 23-28 kDa GTP-binding proteins were present in LDM in approximately the same concentrations as in PM. Insulin gave rise to the characteristic translocation of glucose transporters, but failed to alter the subcellular distribution of any of the GTP-binding proteins. Fractionation of the LDM on a discontinuous sucrose gradient revealed that alpha s and alpha i, as detected with antiserum against a common peptide sequence (alpha common), and the bulk of the 23-28 kDa G-proteins sedimented at different sucrose densities. None of the GTP-binding proteins co-sedimented with glucose transporters. Furthermore, the inhibitory effect of GTP[S] on the reconstituted transport activity was lost in the peak fractions of glucose transporters partially purified on the sucrose gradient. These data indicate that LDM from adipocytes contain several GTP-binding proteins in discrete vesicle populations. However, the intracellular GTP-binding proteins are not tightly associated with the

  14. Image Restoration and Analysis of Influenza Virions Binding to Membrane Receptors Reveal Adhesion-Strengthening Kinetics

    PubMed Central

    Lee, Donald W.; Hsu, Hung-Lun; Bacon, Kaitlyn B.; Daniel, Susan

    2016-01-01

    With the development of single-particle tracking (SPT) microscopy and host membrane mimics called supported lipid bilayers (SLBs), stochastic virus-membrane binding interactions can be studied in depth while maintaining control over host receptor type and concentration. However, several experimental design challenges and quantitative image analysis limitations prevent the widespread use of this approach. One main challenge of SPT studies is the low signal-to-noise ratio of SPT videos, which is sometimes inevitable due to small particle sizes, low quantum yield of fluorescent dyes, and photobleaching. These situations could render current particle tracking software to yield biased binding kinetic data caused by intermittent tracking error. Hence, we developed an effective image restoration algorithm for SPT applications called STAWASP that reveals particles with a signal-to-noise ratio of 2.2 while preserving particle features. We tested our improvements to the SPT binding assay experiment and imaging procedures by monitoring X31 influenza virus binding to α2,3 sialic acid glycolipids. Our interests lie in how slight changes to the peripheral oligosaccharide structures can affect the binding rate and residence times of viruses. We were able to detect viruses binding weakly to a glycolipid called GM3, which was undetected via assays such as surface plasmon resonance. The binding rate was around 28 folds higher when the virus bound to a different glycolipid called GD1a, which has a sialic acid group extending further away from the bilayer surface than GM3. The improved imaging allowed us to obtain binding residence time distributions that reflect an adhesion-strengthening mechanism via multivalent bonds. We empirically fitted these distributions using a time-dependent unbinding rate parameter, koff, which diverges from standard treatment of koff as a constant. We further explain how to convert these models to fit ensemble-averaged binding data obtained by assays such

  15. Neurotensin decreases high affinity [3H]-ouabain binding to cerebral cortex membranes.

    PubMed

    Rosin, Carina; Ordieres, María Graciela López; Arnaiz, Georgina Rodríguez de Lores

    2011-12-10

    Previous work from this laboratory showed the ability of neurotensin to inhibit synaptosomal membrane Na(+), K(+)-ATPase activity, the effect being blocked by SR 48692, a non-peptidic antagonist for high affinity neurotensin receptor (NTS1) [López Ordieres and Rodríguez de Lores Arnaiz 2000; 2001]. To further study neurotensin interaction with Na(+), K(+)-ATPase, peptide effect on high affinity [(3)H]-ouabain binding was studied in cerebral cortex membranes. It was observed that neurotensin modified binding in a dose-dependent manner, leading to 80% decrease with 1 × 10(-4)M concentration. On the other hand, the single addition of 1 × 10(-6)M, 1 × 10(-5)M and 1 × 10(-4)M SR 48692 (Sanofi-Aventis, U.S., Inc.) decreased [(3)H]-ouabain binding (in %) to 87 ± 16; 74 ± 16 and 34 ± 17, respectively. Simultaneous addition of neurotensin and SR 48692 led to additive or synergic effects. Partial NTS2 agonist levocabastine inhibited [(3)H]-ouabain binding likewise. Saturation assays followed by Scatchard analyses showed that neurotensin increased K(d) value whereas failed to modify B(max) value, indicating a competitive type interaction of the peptide at Na(+), K(+)-ATPase ouabain site. At variance, SR 48692 decreased B(max) value whereas it did not modify K(d) value. [(3)H]-ouabain binding was also studied in cerebral cortex membranes obtained from rats injected i. p. 30 min earlier with 100 μg and 250 μg/kg SR 48692. It was observed that the 250 μg/kg SR 48692 dose led to 19% decrease in basal [(3)H]-ouabain binding. After SR 48692 treatments, addition of 1 × 10(-6)M led to additive or synergic effect. Results suggested that [(3)H]-ouabain binding inhibition by neurotensin hardly involves NTS1 receptor.

  16. 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.

  17. 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.

  18. [3H]-verapamil binding to rat cardiac sarcolemmal membrane fragments; an effect of ischaemia.

    PubMed Central

    Dillon, J. S.; Nayler, W. G.

    1987-01-01

    The [3H]-verapamil binding activity of rat cardiac sarcolemmal fragments was studied, using membranes harvested from non-perfused, aerobically-perfused and ischaemic hearts. Glass-fibre filters were found to contain specific, high affinity--(KD 38 +/- 3.1 nM) [3H]-verapamil binding sites--making them unsuitable for use in [3H]-verapamil binding studies. Incubation of membranes from non-perfused hearts in a medium containing 150 mM NaCl, 1 mM CaCl2 and 50 mM Tris revealed two populations of [3H]-verapamil binding sites. When centrifugation instead of filtration was used to separate bound and free [3H]-verapamil, high affinity sites with a KD of 0.57 +/- 0.19 microM and a Bmax of 38 +/- 5.2 pmol mg-1 protein, and low affinity sites with a KD of 78 +/- 27.5 microM and a Bmax of 2.9 +/- 1.3 nmol mg-1 protein were detected. However, only low affinity binding sites could be detected in membranes which had been incubated in a cation-free medium containing 50 mM Tris. [3H]-verapamil binding to the low and high affinity sites was saturable, reversible, stereospecific and displaceable by D600 greater than diltiazem greater than Ca2+ but not by nifedipine, nitrendipine, nisoldipine or prazosin. The two populations of binding sites survived aerobic perfusion and 60 min ischaemia at 37 degrees C. Ischaemia reduced the Bmax and KD but selectivity was maintained. PMID:3028561

  19. Anionic Lipids: Determinants of Binding Cytotoxins from Snake Venom on the Surface of Cell Membranes

    PubMed Central

    Boldyrev, I.A.; Omelkov, A.V.; Utkin, Yu.N.; Efremov, R.G.

    2010-01-01

    The cytotoxic properties of cytotoxins (CTs) from snake venom are mediated by their interaction with the cell membrane. The hydrophobic pattern containing the tips of loops I–III and flanked by polar residues is known to be a membrane–binding motif of CTs. However, this is not enough to explain the difference in activity among various CTs which are similar in sequence and in 3D structure. The mechanism of further CT–membrane interaction leading to pore formation and cell death still remains unknown. Published experimental data on the specific interaction between CT and low molecular weight anionic components (sulphatide) of the bilayer point to the existence of corresponding ligand binding sites on the surface of toxin molecules. In this work we study the membrane–lytic properties of CT I, CT II (Naja oxiana), and Ct 4 (Naja kaouthia), which belong to different structural and functional types (P– and S–type) of CTs, by measuring the intensity of a fluorescent dye, calcein released from liposomes containing a phosphatidylserine (PS) lipid as an anionic component. Using molecular docking simulations, we find and characterize three sites in CT molecules that can potentially bind the PS polar head. Based on the data obtained, we suggest a hypothesis that CTs can specifically interact with one or more of the anionic lipids (in particular, with PS) contained in the membrane, thus facilitating the interaction between CTs and the lipid bilayer of a cell membrane. PMID:22649646

  20. Solution structure and membrane binding of the toxin fst of the par addiction module.

    PubMed

    Göbl, Christoph; Kosol, Simone; Stockner, Thomas; Rückert, Hanna M; Zangger, Klaus

    2010-08-10

    The par toxin-antitoxin system is required for the stable inheritance of the plasmid pAD1 in its native host Enterococcus faecalis. It codes for the toxin Fst and a small antisense RNA which inhibits translation of toxin mRNA, and it is the only known antisense regulated toxin-antitoxin system in Gram-positive bacteria. This study presents the structure of the par toxin Fst, the first atomic resolution structure of a component of an antisense regulated toxin-antitoxin system. The mode of membrane binding was determined by relaxation enhancements in a paramagnetic environment and molecular dynamics simulation. Fst forms a membrane-binding alpha-helix in the N-terminal part and contains an intrinsically disordered region near the C-terminus. It binds in a transmembrane orientation with the C-terminus likely pointing toward the cytosol. Membrane-bound, alpha-helical peptides are frequently found in higher organisms as components of the innate immune system. Despite similarities to these antimicrobial peptides, Fst shows neither hemolytic nor antimicrobial activity when applied externally to a series of bacteria, fungal cells, and erythrocytes. Moreover, its charge distribution, orientation in the membrane, and structure distinguish it from antimicrobial peptides.

  1. 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.

  2. The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein

    PubMed Central

    Qin, Wenxia; Wunderley, Lydia; Barrett, Anne L.; High, Stephen; Woodman, Philip G.

    2016-01-01

    LITAF (LPS-induced TNF-activating factor) is an endosome-associated integral membrane protein important for multivesicular body sorting. Several mutations in LITAF cause autosomal-dominant Charcot Marie Tooth disease type 1C. These mutations map to a highly conserved C-terminal region, termed the LITAF domain, which includes a 22 residue hydrophobic sequence and flanking cysteine-rich regions that contain peptide motifs found in zinc fingers. Although the LITAF domain is thought to be responsible for membrane integration, the membrane topology of LITAF has not been established. Here, we have investigated whether LITAF is a tail-anchored (TA) membrane-spanning protein or monotopic membrane protein. When translated in vitro, LITAF integrates poorly into ER-derived microsomes compared with Sec61β, a bona fide TA protein. Furthermore, introduction of N-linked glycosylation reporters shows that neither the N-terminal nor C-terminal domains of LITAF translocate into the ER lumen. Expression in cells of an LITAF construct containing C-terminal glycosylation sites confirms that LITAF is not a TA protein in cells. Finally, an immunofluorescence-based latency assay showed that both the N- and C-termini of LITAF are exposed to the cytoplasm. Recombinant LITAF contains 1 mol/mol zinc, while mutation of predicted zinc-binding residues disrupts LITAF membrane association. Hence, we conclude that LITAF is a monotopic membrane protein whose membrane integration is stabilised by a zinc finger. The related human protein, CDIP1 (cell death involved p53 target 1), displays identical membrane topology, suggesting that this mode of membrane integration is conserved in LITAF family proteins. PMID:27582497

  3. Membrane Topology and DNA-Binding Ability of the Streptococcal CpsA Protein▿

    PubMed Central

    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 lysozyme to phospholipid bilayers: evidence for protein aggregation upon membrane association.

    PubMed

    Gorbenko, Galyna P; Ioffe, Valeriya M; Kinnunen, Paavo K J

    2007-07-01

    Biological functions of lysozyme, including its antimicrobial, antitumor, and immune-modulatory activities have been suggested to be largely determined by the lipid binding properties of this protein. To gain further insight into these interactions on a molecular level the association of lysozyme to liposomes composed of either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-phosphatidylserine, or bovine heart cardiolipin was studied by a combination of fluorescence techniques. The characteristics of the adsorption of lysozyme to lipid bilayers were investigated using fluorescein 5'-isothiocyanate labeled protein, responding to membrane association by a decrease in fluorescence. Upon increasing the content of anionic phospholipids in lipid vesicles, the binding isotherms changed from Langmuir-like to sigmoidal. Using adsorption models based on scaled particle and double-layer theories, this finding was rationalized in terms of self-association of the membrane-bound protein. The extent of quenching of lysozyme tryptophan fluorescence by acrylamide decreased upon membrane binding, revealing a conformational transition for the protein upon its surface association, resulting in a diminished access of the fluorophore to the aqueous phase. Steady-state fluorescence anisotropy of bilayer-incorporated probe 1,6-diphenyl-1,3,5-hexatriene was measured at varying lipid-to-protein molar ratios. Lysozyme was found to increase acyl-chain order for liposomes with the content of acidic phospholipid exceeding 10 mol %. Both electrostatic and hydrophobic protein-lipid interactions can be concluded to modulate the aggregation behavior of lysozyme when bound to lipid bilayers. Modulation of lysozyme aggregation propensity by membrane binding may have important implications for protein fibrillogenesis in vivo. Disruption of membrane integrity by the aggregated

  5. Binding of Lysozyme to Phospholipid Bilayers: Evidence for Protein Aggregation upon Membrane Association

    PubMed Central

    Gorbenko, Galyna P.; Ioffe, Valeriya M.; Kinnunen, Paavo K. J.

    2007-01-01

    Biological functions of lysozyme, including its antimicrobial, antitumor, and immune-modulatory activities have been suggested to be largely determined by the lipid binding properties of this protein. To gain further insight into these interactions on a molecular level the association of lysozyme to liposomes composed of either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-phosphatidylserine, or bovine heart cardiolipin was studied by a combination of fluorescence techniques. The characteristics of the adsorption of lysozyme to lipid bilayers were investigated using fluorescein 5′-isothiocyanate labeled protein, responding to membrane association by a decrease in fluorescence. Upon increasing the content of anionic phospholipids in lipid vesicles, the binding isotherms changed from Langmuir-like to sigmoidal. Using adsorption models based on scaled particle and double-layer theories, this finding was rationalized in terms of self-association of the membrane-bound protein. The extent of quenching of lysozyme tryptophan fluorescence by acrylamide decreased upon membrane binding, revealing a conformational transition for the protein upon its surface association, resulting in a diminished access of the fluorophore to the aqueous phase. Steady-state fluorescence anisotropy of bilayer-incorporated probe 1,6-diphenyl-1,3,5-hexatriene was measured at varying lipid-to-protein molar ratios. Lysozyme was found to increase acyl-chain order for liposomes with the content of acidic phospholipid exceeding 10 mol %. Both electrostatic and hydrophobic protein-lipid interactions can be concluded to modulate the aggregation behavior of lysozyme when bound to lipid bilayers. Modulation of lysozyme aggregation propensity by membrane binding may have important implications for protein fibrillogenesis in vivo. Disruption of membrane integrity by the aggregated

  6. Dynamic interactions between a membrane binding protein and lipids induce fluctuating diffusivity

    PubMed Central

    Yamamoto, Eiji; Akimoto, Takuma; Kalli, Antreas C.; Yasuoka, Kenji; Sansom, Mark S. P.

    2017-01-01

    Pleckstrin homology (PH) domains are membrane-binding lipid recognition proteins that interact with phosphatidylinositol phosphate (PIP) molecules in eukaryotic cell membranes. Diffusion of PH domains plays a critical role in biological reactions on membrane surfaces. Although diffusivity can be estimated by long-time measurements, it lacks information on the short-time diffusive nature. We reveal two diffusive properties of a PH domain bound to the surface of a PIP-containing membrane using molecular dynamics simulations. One is fractional Brownian motion, attributed to the motion of the lipids with which the PH domain interacts. The other is temporally fluctuating diffusivity; that is, the short-time diffusivity of the bound protein changes substantially with time. Moreover, the diffusivity for short-time measurements is intrinsically different from that for long-time measurements. This fluctuating diffusivity results from dynamic changes in interactions between the PH domain and PIP molecules. Our results provide evidence that the complexity of protein-lipid interactions plays a crucial role in the diffusion of proteins on biological membrane surfaces. Changes in the diffusivity of PH domains and related membrane-bound proteins may in turn contribute to the formation/dissolution of protein complexes in membranes. PMID:28116358

  7. 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.

  8. Structural Thermodynamics of myr-Src(2–19) Binding to Phospholipid Membranes

    PubMed Central

    Scheidt, Holger A.; Klingler, Johannes; Huster, Daniel; Keller, Sandro

    2015-01-01

    Many proteins are anchored to lipid bilayer membranes through a combination of hydrophobic and electrostatic interactions. In the case of the membrane-bound nonreceptor tyrosine kinase Src from Rous sarcoma virus, these interactions are mediated by an N-terminal myristoyl chain and an adjacent cluster of six basic amino-acid residues, respectively. In contrast with the acyl modifications of other lipid-anchored proteins, the myristoyl chain of Src does not match the host lipid bilayer in terms of chain conformation and dynamics, which is attributed to a tradeoff between hydrophobic burial of the myristoyl chain and repulsion of the peptidic moiety from the phospholipid headgroup region. Here, we combine thermodynamic information obtained from isothermal titration calorimetry with structural data derived from 2H, 13C, and 31P solid-state nuclear magnetic resonance spectroscopy to decipher the hydrophobic and electrostatic contributions governing the interactions of a myristoylated Src peptide with zwitterionic and anionic membranes made from lauroyl (C12:0) or myristoyl (C14:0) lipids. Although the latter are expected to enable better hydrophobic matching, the Src peptide partitions more avidly into the shorter-chain lipid analog because this does not require the myristoyl chain to stretch extensively to avoid unfavorable peptide/headgroup interactions. Moreover, we find that Coulombic and intrinsic contributions to membrane binding are not additive, because the presence of anionic lipids enhances membrane binding more strongly than would be expected on the basis of simple Coulombic attraction. PMID:26244740

  9. Coordinated autoinhibition of F-BAR domain membrane binding and WASp activation by Nervous Wreck

    PubMed Central

    Stanishneva-Konovalova, Tatiana B.; Kelley, Charlotte F.; Eskin, Tania L.; Messelaar, Emily M.; Wasserman, Steven A.; Sokolova, Olga S.

    2016-01-01

    Membrane remodeling by Fes/Cip4 homology-Bin/Amphiphysin/Rvs167 (F-BAR) proteins is regulated by autoinhibitory interactions between their SRC homology 3 (SH3) and F-BAR domains. The structural basis of autoregulation, and whether it affects interactions of SH3 domains with other cellular ligands, remain unclear. Here we used single-particle electron microscopy to determine the structure of the F-BAR protein Nervous Wreck (Nwk) in both soluble and membrane-bound states. On membrane binding, Nwk SH3 domains do not completely dissociate from the F-BAR dimer, but instead shift from its concave surface to positions on either side of the dimer. Unexpectedly, along with controlling membrane binding, these autoregulatory interactions inhibit the ability of Nwk-SH3a to activate Wiskott–Aldrich syndrome protein (WASp)/actin related protein (Arp) 2/3-dependent actin filament assembly. In Drosophila neurons, Nwk autoregulation restricts SH3a domain-dependent synaptopod formation, synaptic growth, and actin organization. Our results define structural rearrangements in Nwk that control F-BAR–membrane interactions as well as SH3 domain activities, and suggest that these two functions are tightly coordinated in vitro and in vivo. PMID:27601635

  10. Effect of low-dose testosterone treatment on androgen regulated proteins prostate specific antigen and sex hormone binding globulin in short prepubertal boys: lack of initiation of puberty.

    PubMed

    Gupta, M K; Brown, D C; Faiman, C; Kelnar, C J H; Wu, F C W

    2003-01-01

    The efficacy of testosterone undecanoate (TU) treatment in constitutional delay of growth (CHD) is well recognized. We investigated its role in initiating puberty. Sera taken prior to, just after 6 months on and after 6 months off treatment with TU (20 mg daily) were analyzed from eight boys and compared to results from eight boys receiving placebo. Prostate specific antigen (PSA) and sex hormone binding globulin (SHBG), sleep-entrained pulsatility and mean overnight luteinizing hormone (mLH), and morning testosterone (T) levels were measured. Free androgen index (FAI) was calculated. Testicular volume (TV) and growth parameters were assessed. During treatment, there was a significant increase in height velocity in boys taking TU vs placebo (mean +/- SD: 5.7 +/- 2.0 vs 3.2 +/- 0.9 cm/year, p = 0.008) but no significant differences were observed in regard to LH pulsatility, mLH, T, SHBG, FAI, PSA and TV values. PSA was detectable in four patients (two each in the TU and placebo groups) at 6 months off treatment indicating pubertal progression. Among the hormones measured, only pretreatment mLH levels were significantly higher in the PSA-positive patients compared to 12 PSA-negative patients (mean +/- SEM: 1.5 +/- 0.39 vs 0.37 +/- 0.06 IU/l, p < 0.001). In conclusion, TU treatment shows no significant effect on initiation or advancement of puberty despite its resultant growth acceleration. Among the hormonal changes studied, mLH levels were the earliest indicator of pubertal initiation.

  11. 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

  12. Did androgen-binding protein paralogs undergo neo- and/or Subfunctionalization as the Abp gene region expanded in the mouse genome?

    PubMed

    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.

  13. A membrane cytoskeleton from Dictyostelium discoideum. I. Identification and partial characterization of an actin-binding activity

    PubMed Central

    1981-01-01

    Dictyostelium discoideum plasma membranes isolated by each of three procedures bind F-actin. The interactions between these membranes and actin are examined by a novel application of falling ball viscometry. Treating the membranes as multivalent actin-binding particles analogous to divalent actin-gelation factors, we observe large increases in viscosity (actin cross-linking) when membranes of depleted actin and myosin are incubated with rabbit skeletal muscle F-actin. Pre- extraction of peripheral membrane proteins with chaotropes or the inclusion of Triton X-100 during the assay does not appreciably diminish this actin cross-linking activity. Lipid vesicles, heat- denatured membranes, proteolyzed membranes, or membranes containing endogenous actin show minimal actin cross-linking activity. Heat- denatured, but not proteolyzed, membranes regain activity when assayed in the presence of Triton X-100. Thus, integral membrane proteins appear to be responsible for some or all of the actin cross-linking activity of D. discoideum membranes. In the absence of MgATP, Triton X- 100 extraction of isolated D. discoideum membranes results in a Triton- insoluble residue composed of actin, myosin, and associated membrane proteins. The inclusion of MgATP before and during Triton extraction greatly diminishes the amount of protein in the Triton-insoluble residue without appreciably altering its composition. Our results suggest the existence of a protein complex stabilized by actin and/or myosin (membrane cytoskeleton) associated with the D. discoideum plasma membrane. PMID:6894148

  14. Transcriptional network of androgen receptor in prostate cancer progression.

    PubMed

    Takayama, Ken-ichi; Inoue, Satoshi

    2013-08-01

    The androgen receptor belongs to the nuclear receptor superfamily and functions as a ligand-dependent transcription factor. It binds to the androgen responsive element and recruits coregulatory factors to modulate gene transcription. In addition, the androgen receptor interacts with other transcription factors, such as forkhead box A1, and other oncogenic signaling pathway molecules that bind deoxyribonucleic acid and regulate transcription. Androgen receptor signaling plays an important role in the development of prostate cancer. Prostate cancer cells proliferate in an androgen-dependent manner, and androgen receptor blockade is effective in prostate cancer therapy. However, patients often progress to castration-resistant prostate cancer with elevated androgen receptor expression and hypersensitivity to androgen. Recently, comprehensive analysis tools, such as complementary DNA microarray, chromatin immunoprecipitation-on-chip and chromatin immunoprecipitation-sequence, have described the androgen-mediated diverse transcriptional program and gene networks in prostate cancer. Furthermore, functional and clinical studies have shown that some of the androgen receptor-regulated genes could be prognostic markers and potential therapeutic targets for the treatment of prostate cancer, particularly castration-resistant prostate cancer. Thus, identifying androgen receptor downstream signaling events and investigating the regulation of androgen receptor activity is critical for understanding the mechanism of carcinogenesis and progression to castration-resistant prostate cancer.

  15. Separate [3H]-nitrendipine binding sites in mitochondria and plasma membranes of bovine adrenal medulla.

    PubMed Central

    Ballesta, J. J.; Garcia, A. G.; Gutierrez, L. M.; Hidalgo, M. J.; Palmero, M.; Reig, J. A.; Viniegra, S.

    1990-01-01

    1. Two binding sites for the 1,4-dihydropyridine (DHP) derivative [3H]-nitrendipine have been found in the bovine adrenal medulla. The high-affinity site (Kd = 0.48 nM and Bmax = 128 fmol mg-1 protein) was specifically located in purified plasma membranes. The low-affinity site (Kd = 252 nM and Bmax = 169 pmol mg-1 protein) was located only in mitochondria. Chromaffin granule membranes lacked specific binding sites for [3H]-nitrendipine. 2. Kinetic analysis of the rates of association and dissociation of [3H]-nitrendipine, saturation isotherms and displacement experiments with unlabelled nitrendipine and PN200-110 revealed single, homogeneous populations of high- and low-affinity sites in plasma and mitochondrial membranes, respectively. 3. The high affinity site was sensitive to Ca2+ deprivation and heating; it was practically unaffected by changes in ionic strength of the medium and its optimal pH was slightly alkaline. This site exhibited a strong DHP stereoselectivity; diltiazem increased and verapamil decreased the affinity of [3H]-nitrendipine. 4. In contrast, binding of [3H]-nitrendipine to the low affinity site was more heat resistant and less affected by Ca2+ removal. Its optimal pH was slightly acid and the increase in ionic strength enhanced the number of available sites. The site had no DHP stereoselectivity. Verapamil decreased the dissociation constant of [3H]-nitrendipine acting in a non-competitive manner; diltiazem did not affect equilibrium binding parameters of [3H]-nitrendipine. 5. These results suggest that both biding sites reflect different receptor entities. The high-affinity binding site corresponds to the dihydropyridine receptor associated with the L-type calcium channel. The function of the mitochondrial, low-affinity binding site is, at present, unknown. PMID:1704272

  16. Arabidopsis Membrane Steroid Binding Protein 1 Is Involved in Inhibition of Cell ElongationW⃞

    PubMed Central

    Yang, Xiao-Hua; Xu, Zhi-Hong; Xue, Hong-Wei

    2005-01-01

    A putative Membrane Steroid Binding Protein (designated MSBP1) was identified and functionally characterized as a negative regulator of cell elongation in Arabidopsis thaliana. The MSBP1 gene encodes a 220–amino acid protein that can bind to progesterone, 5-dihydrotestosterone, 24-epi-brassinolide (24-eBL), and stigmasterol with different affinities in vitro. Transgenic plants overexpressing MSBP1 showed short hypocotyl phenotype and increased steroid binding capacity in membrane fractions, whereas antisense MSBP1 transgenic plants showed long hypocotyl phenotypes and reduced steroid binding capacity, indicating that MSBP1 negatively regulates hypocotyl elongation. The reduced cell elongation of MSBP1-overexpressing plants was correlated with altered expression of genes involved in cell elongation, such as expansins and extensins, indicating that enhanced MSBP1 affected a regulatory pathway for cell elongation. Suppression or overexpression of MSBP1 resulted in enhanced or reduced sensitivities, respectively, to exogenous progesterone and 24-eBL, suggesting a negative role of MSBP1 in steroid signaling. Expression of MSBP1 in hypocotyls is suppressed by darkness and activated by light, suggesting that MSBP1, as a negative regulator of cell elongation, plays a role in plant photomorphogenesis. This study demonstrates the functional roles of a steroid binding protein in growth regulation in higher plants. PMID:15608331

  17. 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

  18. Alterations in Ca2+ binding by and composition of the cardiac sarcolemmal membrane in chronic diabetes.

    PubMed Central

    Pierce, G N; Kutryk, M J; Dhalla, N S

    1983-01-01

    Chronic streptozotocin-induced diabetes in rats was associated with a significant loss in the ability of isolated cardiac sarcolemmal membranes to bind Ca2+. Administration of insulin to the diabetic rats normalized the sarcolemmal Ca2+ binding capacity. The content of sialic acid residues, which are considered to represent a superficial Ca2+ pool in sarcolemma, was decreased in preparations from diabetic rats, and this change also was reversible upon insulin treatment of the diabetic rats. Treatment of sarcolemma with neuraminidase decreased Ca2+ binding by 37% in control preparations but had no effect on diabetic preparations. Diphosphatidylglycerol content was decreased but other acidic phospholipids such as phosphatidylinositol and phosphatidylserine, which also bind Ca2+, were not altered during diabetes. An increase in lysophosphatidylcholine and a decrease in phosphatidylethanolamine contents were observed in membranes isolated from diabetic rats. These results suggest that some alterations occur in Ca2+ binding and composition of heart sarcolemma in chronically diabetic rats and may provide further insight into the pathogenesis of diabetic cardiomyopathy. PMID:6577435

  19. Novel androgen receptor gene mutation in patient with complete androgen insensitivity syndrome.

    PubMed

    Ning, Ye; Zhang, Feng; Zhu, Yong; Chen, Huixing; Lu, Jianqi; Li, Zheng

    2012-07-01

    To present a rare case of a patient probably with complete androgen insensitivity syndrome (CAIS) and studied its potential genetic cause. A 24-year-old woman with a normal-appearing vulva and vagina presented to us because of primary amenorrhea. Imaging studies showed no uterus or ovary development but inguinal cryptorchism. Histopathologic examination revealed normal testicular structures. Sequencing the CAIS-associated androgen receptor gene revealed a novel missense mutation of T to G (F698L). A novel androgen receptor gene mutation in the ligand binding domain was detected in the present patient with CAIS, supporting the important role of an androgen receptor defect in the etiology of CAIS.

  20. Modulation of [3H]diazepam binding in rat cortical membranes by GABAA agonists.

    PubMed

    Wong, E H; Iversen, L L

    1985-04-01

    GABAA receptor agonists modulate [3H]diazepam binding in rat cortical membranes with different efficacies. At 23 degrees C, the relative potencies for enhancement of [3H]diazepam binding by agonists parallel their potencies in inhibiting [3H]gamma-aminobutyric acid [( 3H]GABA) binding. The agonist concentrations needed for enhancement of [3H]diazepam binding are up to 35 times higher than for [3H]GABA binding and correspond closely to the concentrations required for displacement of [3H]bicuculline methochloride (BMC) binding. The maximum enhancement of [3H]diazepam varied among agonists: muscimol = GABA greater than isoguvacine greater than 3-aminopropane sulphonic acid (3APS) = imidazoleacetic acid (IAA) greater than 4,5,6,7-tetrahydroisoxazolo (4,5,6)-pyridin-3-ol (THIP) = taurine greater than piperidine 4-sulphonic acid (P4S). At 37 degrees C, the potencies of agonists remained unchanged, but isoguvacine, 3 APS, and THIP acquired efficacies similar to GABA, whereas IAA, taurine, and P4S maintained their partial agonist profiles. At both temperatures the agonist-induced enhancement of [3H]diazepam binding was reversible by bicuculline methobromide and by the steroid GABA antagonist RU 5135. These results stress the importance of studying receptor-receptor interaction under near-physiological conditions and offer an in vitro assay that may predict the agonist status of putative GABA receptor ligands.

  1. 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.

  2. Specific membrane binding of factor VIII is mediated by O-phospho-L-serine, a moiety of phosphatidylserine.

    PubMed

    Gilbert, G E; Drinkwater, D

    1993-09-21

    Phosphatidylserine, a negatively charged lipid, is exposed on the platelet membrane following cell stimulation, correlating with the expression of factor VIII receptors. We have explored the importance of the negative electrostatic potential of phosphatidylserine vs chemical moieties of phosphatidylserine for specific membrane binding of factor VIII. Fluorescein-labeled factor VIII bound to membranes containing 15% phosphatidic acid, a negatively charged phospholipid, with low affinity compared to phosphatidylserine-containing membranes. Binding was not specific as it was inhibited by other proteins in plasma. Factor VIII bound to membranes containing 10% phosphatidylserine in spite of a varying net charge provided by 0-15% stearylamine, a positively charged lipid. The soluble phosphatidylserine moiety, O-phospho-L-serine, inhibited factor VIII binding to phosphatidylserine-containing membranes with a Ki of 20 mM, but the stereoisomer, O-phospho-D-serine, was 5-fold less effective. Furthermore, binding of factor VIII to membranes containing synthetic phosphatidyl-D-serine was 5-fold less than binding to membranes containing phosphatidyl-L-serine. Membranes containing synthetic phosphatidyl-L-homoserine, differing from phosphatidylserine by a single methylene, supported high-affinity binding, but it was not specific as factor VIII was displaced by other plasma proteins. O-Phospho-L-serine also inhibited the binding of factor VIII to platelet-derived microparticles with a Ki of 20 mM, and the stereoisomer was 4-fold less effective. These results indicate that membrane binding of factor VIII is mediated by a stereoselective recognition O-phospho-L-serine of phosphatidylserine and that negative electrostatic potential is of lesser importance.

  3. Membrane-proximal binding of STAT3 revealed by cancer-associated receptor variants.

    PubMed

    Ulaganathan, Vijay K; Ullrich, Axel

    2016-05-01

    In cancer biology, somatic mutations in the extracellular (ligand binding) and cytosolic (functional/catalytic) domains are pursued with great interest. However, in our recent publication we report that germline mutations in the membrane-proximal region of type I receptors are able to modulate the amplitude of signal transducer and activator of transcription 3 (STAT3) signaling in cells. This unexpected finding has implications for the prognosis of heritable cancer.

  4. 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

  5. Structural feature extraction protocol for classifying reversible membrane binding protein domains.

    PubMed

    Källberg, Morten; Lu, Hui

    2009-01-01

    Machine learning based classification protocols for automated function annotation of protein structures have in many instances proven superior to simpler sequence based procedures. Here we present an automated method for extracting features from protein structures by construction of surface patches to be used in such protocols. The utility of the developed patch-growing procedure is exemplified by its ability to identify reversible membrane binding domains from the C1, C2, and PH families.

  6. 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

  7. Alpha-synuclein binds to the inner membrane of mitochondria in an α-helical conformation.

    PubMed

    Robotta, Marta; Gerding, Hanne R; Vogel, Antonia; Hauser, Karin; Schildknecht, Stefan; Karreman, Christiaan; Leist, Marcel; Subramaniam, Vinod; Drescher, Malte

    2014-11-24

    The human alpha-Synuclein (αS) protein is of significant interest because of its association with Parkinson's disease and related neurodegenerative disorders. The intrinsically disordered protein (140 amino acids) is characterized by the absence of a well-defined structure in solution. It displays remarkable conformational flexibility upon macromolecular interactions, and can associate with mitochondrial membranes. Site-directed spin-labeling in combination with electron paramagnetic resonance spectroscopy enabled us to study the local binding properties of αS on artificial membranes (mimicking the inner and outer mitochondrial membranes), and to evaluate the importance of cardiolipin in this interaction. With pulsed, two-frequency, double-electron electron paramagnetic resonance (DEER) approaches, we examined, to the best of our knowledge for the first time, the conformation of αS bound to isolated mitochondria.

  8. Identification of 5-hydroxytryptamine1D binding sites in sheep caudate nucleus membranes.

    PubMed

    Pauwels, P J; Palmier, C; Briley, M

    1993-08-03

    Radioligand binding measurements were performed in membranes of sheep caudate nucleus using [3H]5-hydroxytryptamine (5-HT). [3H]5-HT labeled a population of high affinity binding sites with a Kd of 1.9 +/- 0.1 nM and a Bmax of 19.8 +/- 2.2 fmol/mg tissue. Combined 5-HTID/E binding sites were the predominant 5-HT1 subtype, accounting for 78% of the total population of 5-HT1 binding sites. 5-Carboxamidotryptamine (5-CT) and sumatriptan yielded inhibition curves which best fitted a two-site model with high affinity values of 0.8 and 10.1 nM, and 1000 and 206 nM for their low affinity components. The proportion of the high affinity 5-CT and sumatriptan binding sites was 79 and 72%. The binding affinity profile of 5-HT1D binding sites [5-CT > 5-HT > d-LSD > 5-MeOT > sumatriptan > RU 24,969 > metergoline > tryptamine = rauwolscine = methylsergide > yohimbine = methiothepin > TFMPP = 8-OH-DPAT > 2-methyl-5-HT > mCPP = quipazine = CP 93,129 > ketanserin > (-)-propranolol = haloperidol = ipsapirone] compares well to that reported for 5-HT1D receptor sites in human caudate and cortex (correlation coefficient: 0.99 and 0.98). The present results indicate that sheep caudate nucleus is a valid tissue for studying interaction of compounds with 5-HT1D binding sites in the relative absence of 5-HT1E binding sites.

  9. 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.

  10. Assigning membrane binding geometry of cytochrome C by polarized light spectroscopy.

    PubMed

    Caesar, Christina E B; Esbjörner, Elin K; Lincoln, Per; Nordén, Bengt

    2009-04-22

    In this work we demonstrate how polarized light absorption spectroscopy (linear dichroism (LD)) analysis of the peptide ultraviolet-visible spectrum of a membrane-associated protein (cytochrome (cyt) c) allows orientation and structure to be assessed with quite high accuracy in a native membrane environment that can be systematically varied with respect to lipid composition. Cyt c binds strongly to negatively charged lipid bilayers with a distinct orientation in which its alpha-helical segments are on average parallel to the membrane surface. Further information is provided by the LD of the pi-pi( *) transitions of the heme porphyrin and transitions of aromatic residues, mainly a single tryptophan. A good correlation with NMR data was found, and combining NMR structural data with LD angular data allowed the whole protein to be docked to the lipid membrane. When the redox state of cyt c was changed, distinct variations in the LD spectrum of the heme Soret band were seen corresponding to changes in electronic transition energies; however, no significant change in the overall protein orientation or structure was observed. Cyt c is known to interact in a specific manner with the doubly negatively charged lipid cardiolipin, and incorporation of this lipid into the membrane at physiologically relevant levels was indeed found to affect the protein orientation and its alpha-helical content. The detail in which cyt c binding is described in this study shows the potential of LD spectroscopy using shear-deformed lipid vesicles as a new methodology for exploring membrane protein structure and orientation.

  11. Androgen antagonists in androgen target tissues.

    PubMed

    Tindall, D J; Chang, C H; Lobl, T J; Cunningham, G R

    1984-01-01

    Most antiandrogens appear to act by binding to the androgen receptor and competitively inhibiting the binding of testosterone and cihydrotestosterone to the receptor. Focusing on those compounds which appear to inhibit androgen receptor mediated responses, this review discusses the chemistry of those antiandrogens which have been studied to the extent that their mechanism of action is at least partially understood, outlines the mechanism of androgen action as it is currently understood and suggests how antiandrogens might fit in with this mechanism, indicates the major metabolites of several important antiandrogens, and discusses the clinical applications of several antiandrogens. Cyproterone acetate has been studied extensively as a potential male contraceptive. Although it was recognized that 100 mg of cyproterone acetate per day inhibited spermatogenesis, that dose also reduced libido and potency. Following the administration of 10 or 20 mg of cyproterone acetate per day to 15 males for 26 weeks, the following observations were made: the number of motile sperm was reduced; the quality of their motion was impaired; and the ability of the sperm to penetrate cervical mucus was decreased. Sperm density was also suppressed, but neither it nor sperm motility were inhibited to the extent necessary for contraception. Antiandrogens have been demonstrated to be beneficial in treating 5 clinical syndromes or diseases: acne, seborrhea, hirsutism with or without menstrual abnormalities; precocious puberty; benign prostatic hypertrophy; cancer of the prostate; and sexual deviates. Since 3 of these conditions are very common, effective and safe treatment would have a large market. At this time, antiandrogens are widely used in Europe for treatment of seborrhea, acne, and hirsutism and a large Veterans Administration Cooperative Study in the US was approved but has not yet been funded to compare antiandrogens with other treatments for cancer of the prostate. Studies to assess

  12. 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.

  13. An ATP-binding cassette transporter is a major glycoprotein of sea urchin sperm membranes.

    PubMed

    Mengerink, Kathryn J; Vacquier, Victor D

    2002-10-25

    Sperm are terminally differentiated cells that undergo several membrane-altering events before fusion with eggs. One event, the sea urchin sperm acrosome reaction (AR), is blocked by the lectin wheat germ agglutinin (WGA). In an effort to identify proteins involved in the AR induction, the peptide sequence was obtained from a 220-kDa WGA-binding protein. Degenerate PCR and library screening resulted in the full-length deduced amino acid sequence of an ATP-binding cassette transporter, suABCA. The protein of 1,764 residues has two transmembrane regions, two nucleotide-binding domains, and is most closely related to the human ABC subfamily A member 3 transporter (ABCA3). Sequence analysis suggests a large extracellular loop between transmembrane spanning segments 7 and 8, with five N-linked glycosylation sites. An antibody made to the loop region binds to non-permeabilized cells, supporting that this region is extracellular. suABCA is found in sperm membrane vesicles, it can be solubilized with nonionic detergents, and it shifts from 220 to 200 kDa upon protein:N-glycanase F digestion. suABCA localizes to the entire surface of sperm in a punctate pattern, but is not detected in lipid rafts. Based on its relationship to subfamily A, suABCA is most likely involved in phospholipid or cholesterol transport. This is the first investigation of an ABC transporter in animal sperm.

  14. Lysosomal membrane glycoproteins bind cholesterol and contribute to lysosomal cholesterol export

    PubMed Central

    Li, Jian; Pfeffer, Suzanne R

    2016-01-01

    LAMP1 and LAMP2 proteins are highly abundant, ubiquitous, mammalian proteins that line the lysosome limiting membrane, and protect it from lysosomal hydrolase action. LAMP2 deficiency causes Danon’s disease, an X-linked hypertrophic cardiomyopathy. LAMP2 is needed for chaperone-mediated autophagy, and its expression improves tissue function in models of aging. We show here that human LAMP1 and LAMP2 bind cholesterol in a manner that buries the cholesterol 3β-hydroxyl group; they also bind tightly to NPC1 and NPC2 proteins that export cholesterol from lysosomes. Quantitation of cellular LAMP2 and NPC1 protein levels suggest that LAMP proteins represent a significant cholesterol binding site at the lysosome limiting membrane, and may signal cholesterol availability. Functional rescue experiments show that the ability of human LAMP2 to facilitate cholesterol export from lysosomes relies on its ability to bind cholesterol directly. DOI: http://dx.doi.org/10.7554/eLife.21635.001 PMID:27664420

  15. Routine detection of calcium-binding proteins following their adsorption to nitrocellulose membrane filters

    SciTech Connect

    Hincke, M.T.

    1988-04-01

    A routine semiquantitative procedure which permits soluble calcium-binding proteins to be detected following their adsorption to nitrocellulose membrane filters by liquid scintillation counting of specifically bound /sup 45/Ca is described. Proteins with high affinity for calcium such as calmodulin and troponin can be detected with a detection threshold of about 2 ..mu..g per 400 ..mu..l. Modifications to decrease this limit are feasible and are discussed. This technique should allow calcium-binding proteins of unknown function to be assayed during their purification. It was necessary to treat solutions containing /sup 45/Ca with chelex-100 in order to prevent loss of calcium binding which occurred as the decay product (SC/sup 3 +/) accumulated, suggesting that all studies utilizing /sup 45/Ca as a tracer should evaluate possible interference by this ion.

  16. FERM Domain Phosphoinositide Binding Targets Merlin to the Membrane and Is Essential for Its Growth-Suppressive Function ▿

    PubMed Central

    Mani, Timmy; Hennigan, Robert F.; Foster, Lauren A.; Conrady, Deborah G.; Herr, Andrew B.; Ip, Wallace

    2011-01-01

    The neurofibromatosis type 2 tumor suppressor protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of plasma membrane-actin cytoskeleton linkers. For ezrin, phosphatidylinositol 4,5-bisphosphate (PIP2) binding to the amino-terminal FERM domain is required for its conformational activation, proper subcellular localization, and function, but less is known about the role of phosphoinositide binding for merlin. Current evidence indicates that association with the membrane is important for merlin to function as a growth regulator; however, the mechanisms by which merlin localizes to the membrane are less clear. Here, we report that merlin binds phosphoinositides, including PIP2, via a conserved binding motif in its FERM domain. Abolition of FERM domain-mediated phosphoinositide binding of merlin displaces merlin from the membrane and releases it into the cytosol without altering the folding of merlin. Importantly, a merlin protein whose FERM domain cannot bind phosphoinositide is defective in growth suppression. Retargeting the mutant merlin into the membrane using a dual-acylated amino-terminal decapeptide from Fyn is sufficient to restore the growth-suppressive properties to the mutant merlin. Thus, FERM domain-mediated phosphoinositide binding and membrane association are critical for the growth-regulatory function of merlin. PMID:21402777

  17. 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

  18. 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

  19. 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

  20. The Role of Cationic Group Structure in Membrane Binding and Disruption by Amphiphilic Copolymers

    PubMed Central

    Palermo, Edmund F.; Lee, Dong-Kuk; Ramamoorthy, Ayyalusamy; Kuroda, Kenichi

    2010-01-01

    Cationic, amphiphilic polymers are currently being used as antimicrobial agents which disrupt biomembranes, although their mechanism(s) remain poorly understood. Herein, membrane association and disruption by amphiphilic polymers bearing primary, tertiary, or quaternary ammonium salt groups reveals the role of cationic group structure in the polymer-membrane interaction. The dissociation constants of polymers to liposomes of POPC were obtained by a fluorometric assay, exploiting the environmental sensitivity of dansyl moieties in the polymer end groups. Dye leakage from liposomes and solid-state NMR provided further insights into the polymer-induced membrane disruption. Interestingly, the polymers with primary amine groups induced reorganization of the bilayer structure to align lipid headgroups perpendicular to the membrane. The results showed that polymers bearing primary amines exceed the tertiary and quaternary ammonium counterparts in membrane binding and disrupting abilities. This is likely due to enhanced complexation of primary amines to the phosphate groups in the lipids, through a combination of hydrogen bonding and electrostatic interactions. PMID:21171655

  1. Two stage binding of glucagon to receptors in rat liver plasma membranes

    SciTech Connect

    Wyborski, R.J.; Horwitz, E.M.; Gurd, R.S.

    1986-03-05

    A homogeneous class of noncooperative receptors in isolated rat hepatocytes undergoes a time- and temperature-dependent conformation change with glucagon binding. A comparable system exists in rat liver plasma membranes. Dissociation assays (30/sup 0/C) quantify the number of receptors in each conformational state. Membranes incubated without GTP demonstrated two dissociation rates. The fraction of hormone bound to the high affinity state increases with incubation time to a limiting value. With isolated membranes and a concentration of 0.2 nM ((/sup 125/I)Iodotyrosyl/sup 10/)glucagon, the fraction of the high affinity form is significantly greater than that found in isolated hepatocytes. Previous work without GTP indicated that a lack of cooperativity characterized the liver membrane system. Incubation of membranes with 0.1 mM GTP increases the K/sub D/ as determined by competition assays while the slope factor (.98 +/- 0.04) indicated noncooperativity. Furthermore, in the presence of GTP a significantly greater proportion of receptors is in the low affinity state while in the absence of GTP more are in the high affinity state. The data are consistent with a mechanism by which GTP diminishes the conversion of the low affinity state to the high affinity state.

  2. Megadalton-node assembly by binding of Skb1 to the membrane anchor Slf1.

    PubMed

    Deng, Lin; Kabeche, Ruth; Wang, Ning; Wu, Jian-Qiu; Moseley, James B

    2014-09-01

    The plasma membrane contains both dynamic and static microdomains. Given the growing appreciation of cortical microdomains in cell biology, it is important to determine the organizational principles that underlie assembly of compartmentalized structures at the plasma membrane. The fission yeast plasma membrane is highly compartmentalized by distinct sets of cortical nodes, which control signaling for cell cycle progression and cytokinesis. The mitotic inhibitor Skb1 localizes to a set of cortical nodes that provide spatial control over signaling for entry into mitosis. However, it has been unclear whether these nodes contain other proteins and how they might be organized and tethered to the plasma membrane. Here we show that Skb1 forms nodes by interacting with the novel protein Slf1, which is a limiting factor for node formation in cells. Using quantitative fluorescence microscopy and in vitro assays, we demonstrate that Skb1-Slf1 nodes are megadalton structures that are anchored to the membrane by a lipid-binding region in the Slf1 C-terminus. We propose a mechanism for higher-order node formation by Skb1 and Slf1, with implications for macromolecular assemblies in diverse cell types.

  3. Membrane targeting of TIRAP is negatively regulated by phosphorylation in its phosphoinositide-binding motif

    PubMed Central

    Zhao, Xiaolin; Xiong, Wen; Xiao, Shuyan; Tang, Tuo-Xian; Ellena, Jeffrey F.; Armstrong, Geoffrey S.; Finkielstein, Carla V.; Capelluto, Daniel G. S.

    2017-01-01

    Pathogen-activated Toll-like receptors (TLRs), such as TLR2 and TLR4, dimerize and move laterally across the plasma membrane to phosphatidylinositol (4,5)-bisphosphate-enriched domains. At these sites, TLRs interact with the TIR domain-containing adaptor protein (TIRAP), triggering a signaling cascade that leads to innate immune responses. Membrane recruitment of TIRAP is mediated by its phosphoinositide (PI)-binding motif (PBM). We show that TIRAP PBM transitions from a disordered to a helical conformation in the presence of either zwitterionic micelles or monodispersed PIs. TIRAP PBM bound PIs through basic and nonpolar residues with high affinity, favoring a more ordered structure. TIRAP is phosphorylated at Thr28 within its PBM, which leads to its ubiquitination and degradation. We demonstrate that phosphorylation distorts the helical structure of TIRAP PBM, reducing PI interactions and cell membrane targeting. Our study provides the basis for TIRAP membrane insertion and the mechanism by which it is removed from membranes to avoid sustained innate immune responses. PMID:28225045

  4. Intracellular cAMP increases during the positive inotropism induced by androgens in isolated left atrium of rat.

    PubMed

    Velasco, Lucía; Sánchez, Manuel; Rubín, José Manuel; Hidalgo, Agustín; Bordallo, Carmen; Cantabrana, Begoña

    2002-03-01

    Molecular interactions of androgens with the plasma membrane may produce rapid cardiovascular effects that cannot be explained by the classic genomic mechanisms. In this sense, 5 alpha- and 5 beta-dihydrotestosterone-induced an acute positive inotropic effect in isolated left atrium of rat, an effect which may be due to cAMP-dependent mechanisms. To prove this, intracellular levels of cAMP, after exposure to androgens in the organ bath, and binding to beta(1)-adrenoceptors were evaluated. After a 4-min exposure, 5 alpha- and 5 beta-dihydrotestosterone increased cAMP levels from 3.83+/-0.61 to 6.15+/-1.1 and 11.18+/-2.4 pmol cAMP/mg of protein, respectively. These increases were inhibited by atenolol and not modified by treatment of the rats with reserpine. The androgen-induced cAMP increase seems to be produced via an extracellular interaction, because positive inotropism and raised levels of cAMP were produced by 5 alpha-dihydrotestosterone conjugated with bovine serum albumin (BSA). In addition, it is independent of beta(1)-adrenoceptor activation, because neither androgen displaced [(3)H]dihydroalprenolol binding. Therefore, the androgens induced a positive inotropic effect via a postsynaptic effect that increases intracellular levels of cAMP. This effect is modulated by transcriptional mechanisms or by a protein with a short half-life.

  5. 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

    2014-09-07

    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.

  6. Histo-Blood Group Antigen Presentation Is Critical for Binding of Norovirus VLP to Glycosphingolipids in Model Membranes.

    PubMed

    Nasir, Waqas; Frank, Martin; Kunze, Angelika; Bally, Marta; Parra, Francisco; Nyholm, Per-Georg; Höök, Fredrik; Larson, Göran

    2017-03-27

    Virus entry depends on biomolecular recognition at the surface of cell membranes. In the case of glycolipid receptors, these events are expected to be influenced by how the glycan epitope close to the membrane is presented to the virus. This presentation of membrane-associated glycans is more restricted than that of glycans in solution, particularly because of orientational constraints imposed on the glycolipid through its lateral interactions with other membrane lipids and proteins. We have developed and employed a total internal reflection fluorescence microscopy-based binding assay and a scheme for molecular dynamics (MD) membrane simulations to investigate the consequences of various glycan presentation effects. The system studied was histo-blood group antigen (HBGA) epitopes of membrane-bound glycosphingolipids (GSLs) derived from small intestinal epithelium of humans (type 1 chain) and dogs (type 2 chain) interacting with GII.4 norovirus-like particles. Our experimental results showed strong binding to all lipid-linked type 1 chain HBGAs but no or only weak binding to the corresponding type 2 chain HBGAs. This is in contrast to results derived from STD experiments with free HBGAs in solution where binding was observed for Lewis x. The MD data suggest that the strong binding to type 1 chain glycolipids was due to the well-exposed (1,2)-linked α-l-Fucp and (1,4)-linked α-l-Fucp residues, while the weaker binding or lack of binding to type 2 chain HBGAs was due to the very restricted accessibility of the (1,3)-linked α-l-Fucp residue when the glycolipid is embedded in a phospholipid membrane. Our results not only contribute to a general understanding of protein-carbohydrate interactions on model membrane surfaces, particularly in the context of virus binding, but also suggest a possible role of human intestinal GSLs as potential receptors for norovirus uptake.

  7. 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.

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

    PubMed

    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.

  9. 7alpha,11beta-Dimethyl-19-nortestosterone: a potent and selective androgen response modulator with prostate-sparing properties.

    PubMed

    Cook, C Edgar; Kepler, John A

    2005-02-15

    7alpha,11beta-Dimethyl-19-nortestosterone, made by 1,6-methyl addition to 17beta-acetoxy-11beta-methylestra-4,6-dien-3-one, was a highly potent and selective androgen response modulator, with enhanced androgen receptor binding, androgenic activity and anabolic:androgenic ratio over its two monomethyl homologs.

  10. The androgen receptor gene mutations database.

    PubMed

    Gottlieb, B; Trifiro, M; Lumbroso, R; Vasiliou, D M; Pinsky, L

    1996-01-01

    The current version of the androgen receptor (AR) gene mutations database is described. We have added (if available) data on the androgen binding phenotype of the mutant AR, the clinical phenotype of the affected persons, the family history and whether the pathogenicity of a mutation has been proven. Exonic mutations are now listed in 5'-->3' sequence regardless of type and single base pair changes are presented in codon context. Splice site and intronic mutations are listed separately. The database has allowed us to substantiate and amplify the observation of mutational hot spots within exons encoding the AR androgen binding domain. The database is available from EML (ftp://www.ebi.ac.uk/pub/databases/androgen) or as a Macintosh Filemaker file (MC33@musica.mcgill.ca).

  11. Pirenzepine binding to membrane-bound, solubilized and purified muscarinic receptor subtypes

    SciTech Connect

    Baumgold, J.

    1986-05-01

    Muscarinic receptors were purified to near-homogeneity from bovine cortex, an area rich in the putative M1 subtype, and from bovine pons/medulla, an area rich in the putative M2 subtype. In both cases, the receptors were solubilized in digitonin and purified over an affinity column. Both the cortical and pons/medulla preparations yielded receptor proteins of 70,000 daltons. Pirenzepine binding was deduced from its competition with /sup 3/H-N-methyl scopolamine. The binding of pirenzepine to membrane-bound receptors from cortex was best described by a two site model, with approximately half the sites having a Ki of 6.4 x 10/sup -9/ M and the remaining sites having a Ki of 3.5 x 10/sup -7/ M. Membrane-bound receptors from pons/medulla bound pirenzepine according to a one-site model with a Ki of 1.1 x 10/sup -7/ M. After solubilization the two-site binding of cortical receptors became a one-site binding, Ki = 1.1 x 10/sup -7/M. This value was still five-fold lower than that of soluble receptors from pons/medulla. After purification however the affinity of pirenzepine for the pons/medulla receptor increased so that the two putative subtypes bound pirenzepine with approximately the same affinity. These findings suggest that the different pirenzepine binding characteristics used to define muscarinic receptor subtypes are not inherent in the receptor protein itself but may be due to coupling factors associated with the receptor.

  12. Tunable Membrane Binding of the Intrinsically Disordered Dehydrin Lti30, a Cold-Induced Plant Stress Protein[W

    PubMed Central

    Eriksson, Sylvia K.; Kutzer, Michael; Procek, Jan; Gröbner, Gerhard; Harryson, Pia

    2011-01-01

    Dehydrins are intrinsically disordered plant proteins whose expression is upregulated under conditions of desiccation and cold stress. Their molecular function in ensuring plant survival is not yet known, but several studies suggest their involvement in membrane stabilization. The dehydrins are characterized by a broad repertoire of conserved and repetitive sequences, out of which the archetypical K-segment has been implicated in membrane binding. To elucidate the molecular mechanism of these K-segments, we examined the interaction between lipid membranes and a dehydrin with a basic functional sequence composition: Lti30, comprising only K-segments. Our results show that Lti30 interacts electrostatically with vesicles of both zwitterionic (phosphatidyl choline) and negatively charged phospholipids (phosphatidyl glycerol, phosphatidyl serine, and phosphatidic acid) with a stronger binding to membranes with high negative surface potential. The membrane interaction lowers the temperature of the main lipid phase transition, consistent with Lti30’s proposed role in cold tolerance. Moreover, the membrane binding promotes the assembly of lipid vesicles into large and easily distinguishable aggregates. Using these aggregates as binding markers, we identify three factors that regulate the lipid interaction of Lti30 in vitro: (1) a pH dependent His on/off switch, (2) phosphorylation by protein kinase C, and (3) reversal of membrane binding by proteolytic digest. PMID:21665998

  13. The androgen receptor gene mutations database.

    PubMed

    Patterson, M N; Hughes, I A; Gottlieb, B; Pinsky, L

    1994-09-01

    The androgen receptor gene mutations database is a comprehensive listing of mutations published in journals and meetings proceedings. The majority of mutations are point mutations identified in patients with androgen insensitivity syndrome. Information is included regarding the phenotype, the nature and location of the mutations, as well as the effects of the mutations on the androgen binding activity of the receptor. The current version of the database contains 149 entries, of which 114 are unique mutations. The database is available from EMBL (NetServ@EMBL-Heidelberg.DE) or as a Macintosh Filemaker file (mc33001@musica.mcgill.ca).

  14. Electrochemical potential releases a membrane-bound secretion intermediate of maltose-binding protein in Escherichia coli.

    PubMed Central

    Geller, B L

    1990-01-01

    A secretionary intermediate of the Escherichia coli maltose-binding protein accumulated in the inner membrane when the membrane electrochemical potential was reduced and the cytosolic ATP concentration was normal. The intermediate was mature in size, but maintained a conformation similar to the cytosolic precursor form, and not the mature periplasmic protein, as measured by differences in susceptibility to proteinase K in vitro. The intermediate was located on the periplasmic side of the inner membrane. Restoration of the membrane electrochemical potential resulted in the movement of the intermediate from the inner membrane to the periplasm. In other experiments in which the ATP concentration was reduced by 96% and the electrochemical potential remained normal, no intermediate accumulated. Thus, the final step in the export of maltose-binding protein requires the electrochemical potential of the inner membrane and does not require ATP. Images PMID:2203734

  15. Calcium channel antagonists inhibit the acrosome reaction and bind to plasma membranes of sea urchin sperm.

    PubMed Central

    Kazazoglou, T; Schackmann, R W; Fosset, M; Shapiro, B M

    1985-01-01

    As a prerequisite to fertilization, sea urchin sperm undergo an acrosome reaction that is mediated in part by increased permeability to Ca2+, with an attendant rapid, massive intracellular Ca2+ accumulation. The acrosome reaction is inhibited by Ca2+ channel antagonists, including verapamil, D600, and dihydropyridines such as nitrendipine, nimodipine, and nisoldipine. To examine the interaction of Ca2+ antagonists with sperm, a plasma membrane preparation enriched for Na+,K+-ATPase was isolated from sea urchin sperm. These plasma membranes specifically bound [3H]nitrendipine and [3H]verapamil at concentrations similar to those that inhibit the acrosome reaction. The binding of verapamil was sigmoidal and half-maximal at 1 microM. There was a high specificity in the binding interaction, since by competition binding verapamil, (-)-D600, and (+)-D600 had different relative Kd values, 11, 2.5, and 0.5 microM, respectively. These data suggest that sperm mediate the Ca2+ influx required for induction of the acrosome reaction via Ca2+ channels with properties similar, but not identical, to those of other excitable tissues. Images PMID:3856274

  16. ESCRT-0 assembles as a heterotetrameric complex on membranes and binds multiple ubiquitinylated cargoes simultaneously.

    PubMed

    Mayers, Jonathan R; Fyfe, Ian; Schuh, Amber L; Chapman, Edwin R; Edwardson, J Michael; Audhya, Anjon

    2011-03-18

    The ESCRT machinery consists of multiple protein complexes that collectively participate in the biogenesis of multivesicular endosomes (MVEs). The ESCRT-0 complex is composed of two subunits, Hrs and STAM, both of which can engage ubiquitinylated substrates destined for lysosomal degradation. Here, we conduct a comprehensive analysis of ESCRT-0:ubiquitin interactions using isothermal titration calorimetry and define the affinity of each ubiquitin-binding domain (UBD) within the intact ESCRT-0 complex. Our data demonstrate that ubiquitin binding is non-cooperative between the ESCRT-0 UBDs. Additionally, our findings show that the affinity of the Hrs double ubiquitin interacting motif (DUIM) for ubiquitin is more than 2-fold greater than that of UBDs found in STAM, suggesting that Hrs functions as the major ubiquitin-binding protein in ESCRT-0. In vivo, Hrs and STAM localize to endosomal membranes. To study recombinant ESCRT-0 assembly on lipid bilayers, we used atomic force microscopy. Our data show that ESCRT-0 forms mostly heterodimers and heterotetramers of Hrs and STAM when analyzed in the presence of membranes. Consistent with these findings, hydrodynamic analysis of endogenous ESCRT-0 indicates that it exists largely as a heterotetrameric complex of its two subunits. Based on these data, we present a revised model for ESCRT-0 function in cargo recruitment and concentration at the endosome.

  17. Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

    PubMed Central

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

    2013-01-01

    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

  18. 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.

  19. Membrane properties induced by anionic phospholipids and phosphatidylethanolamine are critical for the membrane binding and catalytic activity of human cytochrome P450 3A4.

    PubMed

    Kim, Keon-Hee; Ahn, Taeho; Yun, Chul-Ho

    2003-12-30

    Human cytochrome P450 (CYP) 3A4, a membrane anchoring protein, is the major CYP enzyme present in both liver and small intestine. The enzyme plays a major role in the metabolism of many drugs and procarcinogens. The roles of individual phospholipids and membrane properties in the catalytic activity, membrane binding, and insertion into the membrane of CYP3A4 are poorly understood. Here we report that the catalytic activity of testosterone 6beta-hydroxylation, membrane binding, and membrane insertion of CYP3A4 increase as a function of anionic phospholipid concentration in the order phosphatidic acid (PA) > phosphatidylserine (PS) in a binary system of phosphatidylcholine (PC)/anionic phospholipid and as a function of phosphatidylethanolamine (PE) content in ternary systems of PC/PE/PA or PC/PE/PS having a fixed concentration of anionic phospholipids. These results suggest that PA and PE might help the binding of CYP3A4 to the membrane and the interaction with NPR. Cytochrome b(5) (b(5)) and apolipoprotein b(5) further enhanced the testosterone 6beta-hydroxylation activities of CYP3A4 in all tested phospholipids vesicles with various compositions. Phospholipid-dependent changes of the CYP3A4 conformation were also revealed by altered Trp fluorescence and CD spectra. We also found that PE induced the formation of anionic phospholipid-enriched domains in ternary systems using extrinsic fluorescent probes incorporated into lipid bilayers. Taken together, it can be suggested that the chemical and physical properties of membranes induced by anionic phospholipids and PE are critical for the membrane binding and catalytic activity of CYP3A4.

  20. Influence of gamma subunit prenylation on association of guanine nucleotide-binding regulatory proteins with membranes.

    PubMed Central

    Muntz, K H; Sternweis, P C; Gilman, A G; Mumby, S M

    1992-01-01

    Two approaches were taken to address the possible role of gamma-subunit prenylation in dictating the cellular distribution of guanine nucleotide-binding regulatory proteins. Prenylation of gamma subunits was prevented by site-directed mutagenesis or by inhibiting the synthesis of mevalonate, the precursor of cellular isoprenoids. When beta or gamma subunits were transiently expressed in COS-M6 simian kidney cells (COS) cells, the proteins were found in the membrane fraction by immunoblotting. Immunofluorescence experiments indicated that the proteins were distributed to intracellular structures in addition to plasma membranes. Replacement of Cys68 of gamma with Ser prevented prenylation of the mutant protein and association of the protein with the membrane fraction of COS cells. Immunoblotting results demonstrated that some of the beta subunits were found in the cytoplasm when coexpressed with the nonprenylated mutant gamma subunit. When Neuro 2A cells were treated with compactin to inhibit protein prenylation, a fraction of endogenous beta and gamma was distributed in the cytoplasm. It is concluded that prenylation facilitates association of gamma subunits with membranes, that the cellular location of gamma influences the distribution of beta, and that prenylation is not an absolute requirement for interaction of beta and gamma. Images PMID:1550955

  1. Molecular properties of a novel, hydrophilic cation-binding protein associated with the plasma membrane.

    PubMed

    Ide, Yuki; Nagasaki, Nahoko; Tomioka, Rie; Suito, Momoe; Kamiya, Takehiro; Maeshima, Masayoshi

    2007-01-01

    A new type of protein was found in Arabidopsis thaliana, PCaP1, which is rich in glutamate and lysine residues. The protein bound (45)Ca(2+) even in the presence of a high concentration of Mg(2+). Real-time polymerase chain reaction and histochemical analysis of promoter-beta-glucuronidase fusions revealed that PCaP1 was expressed in most organs. The PCaP1 protein was detected immunochemically in these organs. Treatment of Arabidopsis seedlings with Cu(2+), sorbitol, or flagellin oligopeptide enhanced the transcription. On the other hand, other sugars, abscisic acid, gibberellic acid, dehydration, and low temperature had little or no effect on PCaP1 transcript abundance. The transient expression of PCaP1 fused to green fluorescent protein in Arabidopsis cells and the subcellular fractionation of tissue homogenate showed that PCaP1 protein is localized to the plasma membrane, although PCaP1 has no predicted transmembrane domain. PCaP1 was associated with the plasma membrane under natural conditions and was released from the membrane at high concentrations of Ca(2+) or Mg(2+) in vitro. These results suggest that the hydrophilic protein PCaP1 binds Ca(2+) and other cations and is stably associated with the plasma membrane.

  2. 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

  3. 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

  4. 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.

  5. Using micropatterned lipid bilayer arrays to measure the effect of membrane composition on merocyanine 540 binding.

    PubMed

    Smith, Kathryn A; Conboy, John C

    2011-06-01

    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 binding in 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.

  6. 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.

  7. The GABA agonist THIP a muscimol analogue, does not interfere with the benzodiazepine binding site on rats cortical membranes.

    PubMed

    Maurer, R

    1979-04-01

    THIP, a cyclic analogue of muscimol, is a powerful GABA agonist. It is as active as GABA in displacing [3H]muscimol from its binding site to cerebellar membranes (IC50 = 31.5 +/- 2.5 mM). However, unlike muscimol or GABA, it is devoid of any modulatory interaction with the benzodiazepine binding site on rat's cortical membranes. Homotaurine, isoguvacine and imidazole acetic acid are less active than muscimol and GABA for increasing the affinity of [3H]diazepam to cortical membrane preparations.

  8. Identification of a novel bacterial outer membrane interleukin-1Β-binding protein from Aggregatibacter actinomycetemcomitans.

    PubMed

    Paino, Annamari; Ahlstrand, Tuuli; Nuutila, Jari; Navickaite, Indre; Lahti, Maria; Tuominen, Heidi; Välimaa, Hannamari; Lamminmäki, Urpo; Pöllänen, Marja T; Ihalin, Riikka

    2013-01-01

    Aggregatibacter actinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1β. Some bacterial species can alter their physiological properties as a result of sensing IL-1β. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1β sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1β through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1β than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1β, but this binding was not specific, as a control protein for IL-1

  9. Identification of a Novel Bacterial Outer Membrane Interleukin-1Β-Binding Protein from Aggregatibacter actinomycetemcomitans

    PubMed Central

    Paino, Annamari; Ahlstrand, Tuuli; Nuutila, Jari; Navickaite, Indre; Lahti, Maria; Tuominen, Heidi; Välimaa, Hannamari; Lamminmäki, Urpo; Pöllänen, Marja T.; Ihalin, Riikka

    2013-01-01

    Aggregatibacteractinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1β. Some bacterial species can alter their physiological properties as a result of sensing IL-1β. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1β sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1β through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1β than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1β, but this binding was not specific, as a control protein for IL-1

  10. Interactions of chrysotile and crocidolite asbestos with red blood cell membranes. Chrysotile binds to sialic acid.

    PubMed

    Brody, A R; George, G; Hill, L H

    1983-10-01

    Chrysotile and crocidolite are commonly used forms of asbestos. Hemolysis has been widely used as a test of membrane injury, and it has been shown previously that chrysotile causes rapid breakdown of red blood cells (RBCs), whereas crocidolite is only weakly hemolytic. A reasonable hypothesis set forth to explain the cytotoxic effects of chrysotile maintains that positively charged chrysotile fibers bind to negatively charged sialic acid residues on RBC membranes causing clustering of membrane proteins and increased cell permeability to Na and K ions. Our studies presented here provide two lines of evidence in direct support of this hypothesis. (a) Morphologic--Ultrastructural techniques showed that both chrysotile and crocidolite asbestos bind to and distort more than 85% of RBCs treated for 15 minutes. The distorting effects of chrysotile, but not crocidolite, were almost totally ablated by pretreating the cells with neuraminidase. In addition, gold-conjugated wheat germ agglutinin was used to label the distribution of sialic acid groups on RBC membranes. Pretreatment of the RBCs with chrysotile, but not crocidolite, reduced the number of gold-conjugated wheat germ agglutinin-labeled sites to less than 30% of the control level. (b) Biochemical--The thiobarbituric acid assay was used to determine the percentage of sialic acid that remained with the cell pellet after neuraminidase and/or asbestos treatment. Asbestos treatment alone caused no release of sialic acid from the cells. Neuraminidase treatment for 3.5 hours removed more than 80% of the sialic acid from cell surfaces. Chrysotile, but not crocidolite, asbestos prevented neuraminidase-mediated removal of sialic acid from RBCs. In addition, x-ray energy spectrometry of freeze-dried cells showed that RBCs distorted by chrysotile, but not by crocidolite, exhibited significant alterations in intracellular Na:K ratios. The morphologic and biochemical data strongly support the hypothesis that chrysotile asbestos

  11. Leptospiral Outer Membrane Protein Microarray, a Novel Approach to Identification of Host Ligand-Binding Proteins

    PubMed Central

    Matsunaga, James; Haake, David A.

    2012-01-01

    Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens. PMID:22961849

  12. Conformational Changes Produced by ATP Binding to the Plasma Membrane Calcium Pump*

    PubMed Central

    Mangialavori, Irene C.; Ferreira-Gomes, Mariela S.; Saffioti, Nicolás A.; González-Lebrero, Rodolfo M.; Rossi, Rolando C.; Rossi, Juan Pablo F. C.

    2013-01-01

    The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca2+ with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate. To assess the conformational behavior of the Ca2+ binding domain, we also studied the occlusion of Ca2+, both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca2+ and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E1-E2 model devised using kinetics methodology only. PMID:24025327

  13. Conformational changes produced by ATP binding to the plasma membrane calcium pump.

    PubMed

    Mangialavori, Irene C; Ferreira-Gomes, Mariela S; Saffioti, Nicolás A; González-Lebrero, Rodolfo M; Rossi, Rolando C; Rossi, Juan Pablo F C

    2013-10-25

    The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[(125)I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca(2+) with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate. To assess the conformational behavior of the Ca(2+) binding domain, we also studied the occlusion of Ca(2+), both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca(2+) and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E1-E2 model devised using kinetics methodology only.

  14. 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.

  15. Soluble klotho binds monosialoganglioside to regulate membrane microdomains and growth factor signaling

    PubMed Central

    Dalton, George; An, Sung-Wan; Al-Juboori, Saif I.; Nischan, Nicole; Yoon, Joonho; Dobrinskikh, Evgenia; Hilgemann, Donald W.; Xie, Jian; Luby-Phelps, Kate; Kohler, Jennifer J.; Birnbaumer, Lutz; Huang, Chou-Long

    2017-01-01

    Soluble klotho, the shed ectodomain of the antiaging membrane protein α-klotho, is a pleiotropic endocrine/paracrine factor with no known receptors and poorly understood mechanism of action. Soluble klotho down-regulates growth factor-driven PI3K signaling, contributing to extension of lifespan, cardioprotection, and tumor inhibition. Here we show that soluble klotho binds membrane lipid rafts. Klotho binding to rafts alters lipid organization, decreases membrane’s propensity to form large ordered domains for endocytosis, and down-regulates raft-dependent PI3K/Akt signaling. We identify α2-3-sialyllactose present in the glycan of monosialogangliosides as targets of soluble klotho. α2-3-Sialyllactose is a common motif of glycans. To explain why klotho preferentially targets lipid rafts we show that clustering of gangliosides in lipid rafts is important. In vivo, raft-dependent PI3K signaling is up-regulated in klotho-deficient mouse hearts vs. wild-type hearts. Our results identify ganglioside-enriched lipid rafts to be receptors that mediate soluble klotho regulation of PI3K signaling. Targeting sialic acids may be a general mechanism for pleiotropic actions of soluble klotho. PMID:28069944

  16. 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

  17. 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.

  18. 7alpha-methyl-19-nortestosterone, a synthetic androgen with high potency: structure-activity comparisons with other androgens.

    PubMed

    Kumar, N; Crozat, A; Li, F; Catterall, J F; Bardin, C W; Sundaram, K

    1999-12-31

    CNNT. There was a good correlation between bioactivity and binding affinity to AR for the 7alpha-substituted androgens compared to T. In contrast, relative to their binding affinity to AR, the androgenic potency of DHT and 19-NT was lower compared to T. The reason for the lower in vivo androgenic activity of 19-NT is attributable to its enzymatic conversion to 5alpha-reduced-19-NT in the prostate. In the case of DHT, the lower bioactivity could be attributed to its faster metabolic clearance rate relative to T. The correlation was further investigated in vitro by co-transfection of rat ARcDNA expression plasmid and a reporter plasmid encoding the chloramphenicol acetyl transferase (CAT) gene driven by an androgen inducible promoter into CV-1 cells. All the androgens led to a dose-dependent increase in the CAT activity. MENT was found to be the most potent followed by DHT, 19-NT, T, and CNNT. The specificity of the androgenic response was confirmed by its inhibition with hydroxyflutamide, an antiandrogen. Thus, there was a good correlation between binding affinity and in vitro bioactivity in the transient transfection assay for the androgens. This suggests that the in vivo bioactivity of androgens could be influenced not only by binding affinity to receptors but also by factors such as absorption, binding to serum proteins and metabolism. However, the high potency of MENT is primarily related to its higher affinity to AR.

  19. Non-genomic Actions of Androgens

    PubMed Central

    Foradori, C. D.; Weiser, M. J.; Handa, R. J.

    2008-01-01

    Previous work in the endocrine and neuroendocrine fields has viewed androgen receptors (AR) as a transcription factor activated by testosterone or one of its many metabolites. The bound androgen receptor acts as transcription factor and binds to specific DNA response elements in target gene promoters, causing activation or repression of transcription and subsequently protein synthesis. Over the past two decades evidence has begun to accumulate to implicate androgens, dependent or independent of the AR, in rapid actions at the cellular and organism level. Androgen’s rapid time course of action; effects in the absence or inhibition of the cellular machinery necessary for transcription/translation; and/or the effects of androgens not able to translocate to the nucleus suggest a method of androgen action not initially dependent on genomic mechcanisms (i.e. non-genomic in nature). In the present paper the non-genomic effects of androgens are reviewed, along with a discussion of the possible role non-genomic androgen actions have on animal physiology and behavior. PMID:18093638

  20. Label-free detection of small-molecule binding to a GPCR in the membrane environment.

    PubMed

    Heym, Roland G; Hornberger, Wilfried B; Lakics, Viktor; Terstappen, Georg C

    2015-08-01

    Evaluation of drug-target interaction kinetics is becoming increasingly important during the drug-discovery process to investigate selectivity of a drug and predict in vivo target occupancy. To date, it remains challenging to obtain kinetic information for interactions between G-protein-coupled receptors (GPCRs) and small-molecule ligands in a label-free manner. Often GPCRs need to be solubilized or even stabilized by mutations which can be difficult and is time consuming. In addition, it is often unclear if the native conformation of the receptors is sustained. In this study, surface plasmon resonance (SPR) and surface acoustic wave (SAW) technologies have been used to detect ligand binding to the GPCR chemokine (C-X-C motif) receptor 4 (CXCR4) expressed in lipoparticles. We first evaluated different strategies to immobilize CXCR4-expressing lipoparticles. The highest small-molecule binding signal in SPR and SAW was achieved with a matrix-free carboxymethylated sensor chip coated with wheat germ agglutinin for lipoparticle capturing. Next, the binding kinetics of the anti-CXCR4 antibody 12G5 raised against a conformational epitope (k(on)=1.83×10(6)M(-1)s(-1), k(off)=2.79×10(-4) s(-1)) and the small molecule AMD3100 (k(on)=5.46×10(5)M(-1)s(-1), k(off)=1.01×10(-2)s(-1)) were assessed by SAW. Our kinetic and affinity data are consistent with previously published radioligand-binding experiments using cells and label-free experiments with solubilized CXCR4. This is the first study demonstrating label-free kinetic characterization of small-molecule binding to a GPCR in the membrane environment. The presented method holds the potential to greatly facilitate label-free assay development for GPRCs that can be expressed at high levels in lipoparticles.

  1. Androgen insensitivity syndrome.

    PubMed

    Hughes, Ieuan A; Davies, John D; Bunch, Trevor I; Pasterski, Vickie; Mastroyannopoulou, Kiki; MacDougall, Jane

    2012-10-20

    Androgen insensitivity syndrome in its complete form is a disorder of hormone resistance characterised by a female phenotype in an individual with an XY karyotype and testes producing age-appropriate normal concentrations of androgens. Pathogenesis is the result of mutations in the X-linked androgen receptor gene, which encodes for the ligand-activated androgen receptor--a transcription factor and member of the nuclear receptor superfamily. This Seminar describes the clinical manifestations of androgen insensitivity syndrome from infancy to adulthood, reviews the mechanism of androgen action, and shows examples of how mutations of the androgen receptor gene cause the syndrome. Management of androgen insensitivity syndrome should be undertaken by a multidisciplinary team and include gonadectomy to avoid gonad tumours in later life, appropriate sex-hormone replacement at puberty and beyond, and an emphasis on openness in disclosure.

  2. 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.

  3. A Plant Plasma Membrane ATP Binding Cassette–Type Transporter Is Involved in Antifungal Terpenoid Secretion

    PubMed Central

    Jasiński, Michal; Stukkens, Yvan; Degand, Hervé; Purnelle, Bénédicte; Marchand-Brynaert, Jacqueline; Boutry, Marc

    2001-01-01

    ATP binding cassette (ABC) transporters, which are found in all species, are known mainly for their ability to confer drug resistance. To date, most of the ABC transporters characterized in plants have been localized in the vacuolar membrane and are considered to be involved in the intracellular sequestration of cytotoxins. Working on the assumption that certain ABC transporters might be involved in defense metabolite secretion and their expression might be regulated by the concentration of these metabolites, we treated a Nicotiana plumbaginifolia cell culture with sclareolide, a close analog of sclareol, an antifungal diterpene produced at the leaf surface of Nicotiana spp; this resulted in the appearance of a 160-kD plasma membrane protein, which was partially sequenced. The corresponding cDNA (NpABC1) was cloned and shown to encode an ABC transporter. In vitro and in situ immunodetection showed NpABC1 to be localized in the plasma membrane. Under normal conditions, expression was found in the leaf epidermis. In cell culture and in leaf tissues, NpABC1 expression was strongly enhanced by sclareolide and sclareol. In parallel with NpABC1 induction, cells acquired the ability to excrete a labeled synthetic sclareolide derivative. These data suggest that NpABC1 is involved in the secretion of a secondary metabolite that plays a role in plant defense. PMID:11340184

  4. Upper-body resistance exercise augments vastus lateralis androgen receptor-DNA binding and canonical Wnt/β-catenin signaling compared to lower-body resistance exercise in resistance-trained men without an acute increase in serum testosterone.

    PubMed

    Spillane, Mike; Schwarz, Neil; Willoughby, Darryn S

    2015-06-01

    The purpose of the study was to determine the effect of single bouts of lower-body (LB) and upper- and lower-body (ULB) resistance exercise on serum testosterone concentrations and the effects on muscle testosterone, dihydrotestosterone (DHT), androgen receptor (AR) protein content, and AR-DNA binding. A secondary purpose was to determine the effects on serum wingless-type MMTV integration site (Wnt4) levels and skeletal muscle β-catenin content. In a randomized cross-over design, exercise bouts consisted of a LB and ULB protocol, and each bout was separated by 1 week. Blood and muscle samples were obtained before exercise and 3 and 24h post-exercise; blood samples were also obtained at 0.5, 1, and 2 h post-exercise. Statistical analyses were performed by separate two-way factorial analyses of variance (ANOVA) with repeated measures. No significant differences from baseline were observed in serum total and free testosterone and skeletal muscle testosterone and DHT with either protocol (p>0.05). AR protein was significantly increased at 3 h post-exercise and decreased at 24 h post-exercise for ULB, whereas AR-DNA binding was significantly increased at 3 and 24h post-exercise (p<0.05). In response to ULB, serum Wnt4 was significantly increased at 0.5, 1, and 2 h post-exercise (p<0.05) and β-catenin was significantly increased at 3 and 24 h post-exercise (p<0.05). It was concluded that, despite a lack of increase in serum testosterone and muscle androgen concentrations from either mode of resistance exercise, ULB resistance exercise increased Wnt4/β-catenin signaling and AR-DNA binding.

  5. α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation

    PubMed Central

    Burré, Jacqueline; Sharma, Manu; Südhof, Thomas C.

    2014-01-01

    Physiologically, α-synuclein chaperones soluble NSF attachment protein receptor (SNARE) complex assembly and may also perform other functions; pathologically, in contrast, α-synuclein misfolds into neurotoxic aggregates that mediate neurodegeneration and propagate between neurons. In neurons, α-synuclein exists in an equilibrium between cytosolic and membrane-bound states. Cytosolic α-synuclein appears to be natively unfolded, whereas membrane-bound α-synuclein adopts an α-helical conformation. Although the majority of studies showed that cytosolic α-synuclein is monomeric, it is unknown whether membrane-bound α-synuclein is also monomeric, and whether chaperoning of SNARE complex assembly by α-synuclein involves its cytosolic or membrane-bound state. Here, we show using chemical cross-linking and fluorescence resonance energy transfer (FRET) that α-synuclein multimerizes into large homomeric complexes upon membrane binding. The FRET experiments indicated that the multimers of membrane-bound α-synuclein exhibit defined intermolecular contacts, suggesting an ordered array. Moreover, we demonstrate that α-synuclein promotes SNARE complex assembly at the presynaptic plasma membrane in its multimeric membrane-bound state, but not in its monomeric cytosolic state. Our data delineate a folding pathway for α-synuclein that ranges from a monomeric, natively unfolded form in cytosol to a physiologically functional, multimeric form upon membrane binding, and show that only the latter but not the former acts as a SNARE complex chaperone at the presynaptic terminal, and may protect against neurodegeneration. PMID:25246573

  6. Activation of the Complement Classical Pathway (C1q Binding) by Mesophilic Aeromonas hydrophila Outer Membrane Protein

    PubMed Central

    Merino, Susana; Nogueras, Maria Mercedes; Aguilar, Alicia; Rubires, Xavier; Albertí, Sebastian; Benedí, Vicente Javier; Tomás, Juan M.

    1998-01-01

    The mechanism of killing of Aeromonas hydrophila serum-sensitive strains in nonimmune serum by the complement classical pathway has been studied. The bacterial cell surface component that binds C1q more efficiently was identified as a major outer membrane protein of 39 kDa, presumably the porin II described by D. Jeanteur, N. Gletsu, F. Pattus, and J. T. Buckley (Mol. Microbiol. 6:3355–3363, 1992), of these microorganisms. We have demonstrated that the purified form of porin II binds C1q and activates 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 of factor D). Binding of C1q to other components of the bacterial outer membrane, in particular to rough lipopolysaccharide, could not be demonstrated. Activation of the classical pathway by this lipopolysaccharide was also much less efficient than activation by the outer membrane protein. The strains possessing O-antigen lipopolysaccharide bind less C1q than the serum-sensitive strains, because the outer membrane protein is less accessible, and are resistant to complement-mediated killing. Finally, a similar or identical outer membrane protein (presumably porin II) that binds C1q was shown to be present in strains from the most common mesophilic Aeromonas O serogroups. PMID:9673268

  7. Oncogenic K-Ras Binds to an Anionic Membrane in Two Distinct Orientations: A Molecular Dynamics Analysis.

    PubMed

    Prakash, Priyanka; Zhou, Yong; Liang, Hong; Hancock, John F; Gorfe, Alemayehu A

    2016-03-08

    K-Ras is a membrane-associated GTPase that cycles between active and inactive conformational states to regulate a variety of cell signaling pathways. Somatic mutations in K-Ras are linked to 15-20% of all human tumors. K-Ras attaches to the inner leaflet of the plasma membrane via a farnesylated polybasic domain; however, the structural details of the complex remain poorly understood. Based on extensive (7.5 μs total) atomistic molecular dynamics simulations here we show that oncogenic mutant K-Ras interacts with a negatively charged lipid bilayer membrane in multiple orientations. Of these, two highly populated orientations account for ∼54% of the conformers whose catalytic domain directly interacts with the bilayer. In one of these orientation states, membrane binding involves helices 3 and 4 of the catalytic domain in addition to the farnesyl and polybasic motifs. In the other orientation, β-strands 1-3 and helix 2 on the opposite face of the catalytic domain contribute to membrane binding. Flexibility of the linker region was found to be important for the reorientation. The biological significance of these observations was evaluated by initial experiments in cells overexpressing mutant K-Ras as well as by an analysis of Ras-effector complex structures. The results suggest that only one of the two major orientation states is capable of effector binding. We propose that the different modes of membrane binding may be exploited in structure-based drug design efforts for cancer therapy.

  8. Specific /sup 3/H-DMCM binding to a non-benzodiazepine binding site after silver ion treatment of rat brain membranes

    SciTech Connect

    Honore, T.; Nielsen, M.; Braestrup, C.

    1984-11-26

    Specific binding of the BZ-receptor ligand /sup 3/H-DMCM to rat cortical membranes was dramatically enhanced by preincubation of the homogenate with 0.1 mM silver (Ag/sup +/) nitrate. The binding was completely inhibited by midazolam. Nevertheless, the pharmacological specificity of the Ag/sup +/-enhanced /sup 3/H-DMCM binding was different from that of BZ-receptors. Furthermore, the B/sub max/ value, the regional distribution and the molecular target size determined by radiation inactivation analysis of the Ag/sup +/-enhanced binding site were different from those of BZ-receptors. The results indicate that Ag/sup +/-enhanced /sup 3/H-DMCM binding represent a high affinity metal complex formation between /sup 3/H-DMCM and an unknown brain specific protein of approximately 100,000 daltons molecular weight. 11 references, 3 figures, 4 tables.

  9. 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.

  10. 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.

  11. 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.

  12. Binding of the C-terminal sterile alpha motif (SAM) domain of human p73 to lipid membranes.

    PubMed

    Barrera, Francisco N; Poveda, José A; González-Ros, José M; Neira, José L

    2003-11-21

    The alpha splice variant of p73 (p73alpha), a homologue of the tumor suppressor p53, has close to its C terminus a sterile alpha motif (SAM), SAMp73, that is thought to be involved in protein-protein interactions. Here, we report the lipid binding properties of this domain. Binding was assayed against zwitterionic (phosphatidylcholine) and anionic (phosphatidic acid) lipids and was studied by different biophysical techniques, namely, circular dichroism and fluorescence spectroscopies and differential scanning calorimetry. These techniques unambiguously indicate that SAMp73 binds to lipids. The binding involves protein surface attachment and partial membrane penetration, accompanied by changes in SAMp73 structure.

  13. 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.

  14. Insulin and epidermal growth factor-urogastrone: Affinity crosslinking to specific binding sites in rat liver membranes

    PubMed Central

    Sahyoun, N.; Hock, R. A.; Hollenberg, M. D.

    1978-01-01

    Both insulin and human epidermal growth factor-urogastrone (EGF/URO) can be covalently linked to specific rat liver membrane binding sites by glutaraldehyde coupling followed by sodium borohydride reduction to yield affinity-labeled membrane constituents sufficiently stable for solubilization and further analysis by various techniques. Solubilization of membranes covalently labeled with 125I-labeled insulin yields a component with chromatographic properties identical to those of a soluble insulin receptor characterized in previous studies. A second soluble insulin-binding component that is not revealed by the affinity-labeling method and that has not yet been reported can also be detected. Membranes similarly labeled with 125I-labeled EGF/URO yield one major and two minor ligand-specific soluble (Triton X-100) affinity-labeled components, as detected by chromatography on Sepharose 6B. Further analysis of the EGF/URO-labeled components by affinity chromatography on concanavalin A-Sepharose, by disc gel electrophoresis, and by enzymatic digestion suggests that the major specific binding component for EGF/URO in liver membranes is a glycoprotein subunit of approximately 100,000 daltons that possesses a 20,000-dalton portion inaccessible to proteolytic cleavage when the subunit is anchored in the membrane. The affinity labeling approach described should prove of use for the study of other polypeptide receptors that, like the EGF/URO receptor, lose their ligand recognition property subsequent to membrane solubilization. PMID:205865

  15. The Peripheral Binding of 14-3-3γ to Membranes Involves Isoform-Specific Histidine Residues

    PubMed Central

    Ying, Ming; Halskau, Øyvind; Baumann, Anne; Rodriguez-Larrea, David; Costas, Miguel; Underhaug, Jarl; Sanchez-Ruiz, Jose M.; Martinez, Aurora

    2012-01-01

    Mammalian 14-3-3 protein scaffolds include seven conserved isoforms that bind numerous phosphorylated protein partners and regulate many cellular processes. Some 14-3-3-isoforms, notably γ, have elevated affinity for membranes, which might contribute to modulate the subcellular localization of the partners and substantiate the importance of investigating molecular mechanisms of membrane interaction. By applying surface plasmon resonance we here show that the binding to phospholipid bilayers is stimulated when 14-3-3γ is complexed with its partner, a peptide corresponding to the Ser19-phosphorylated N-terminal region of tyrosine hydroxylase. Moreover, membrane interaction is dependent on salts of kosmotropic ions, which also stabilize 14-3-3γ. Electrostatic analysis of available crystal structures of γ and of the non-membrane-binding ζ-isoform, complemented with molecular dynamics simulations, indicate that the electrostatic potential distribution of phosphopeptide-bound 14-3-3γ is optimal for interaction with the membrane through amphipathic helices at the N-terminal dimerization region. In addition, His158, and especially His195, both specific to 14-3-3γ and located at the convex lateral side, appeared to be pivotal for the ligand induced membrane interaction, as corroborated by site-directed mutagenesis. The participation of these histidine residues might be associated to their increased protonation upon membrane binding. Overall, these results reveal membrane-targeting motifs and give insights on mechanisms that furnish the 14-3-3γ scaffold with the capacity for tuned shuffling from soluble to membrane-bound states. PMID:23189152

  16. 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.

  17. 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

    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.

  18. 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

  19. Modification of L-triiodothyronine binding sites from rat erythrocyte membrane by heating and by proteinase treatments.

    PubMed

    Angel, R C; Botta, J A; Farías, R N

    1987-03-12

    The number of binding sites for L-triiodothyronine in rat erythrocyte membranes was increased 2-fold by incubation at 37 degrees C for 60 min. An increase of approximately 3-fold was found when the incubation was carried out at 50 degrees C. The proteinase inhibitor phenylmethylsulfonyl fluoride abolished the effect. Similar increments in the number of binding sites were obtained by treatment of the membranes with proteinases. The Kd values (0.09 X 10(-10) M and 3.6 X 10(-10) M for the high-affinity and the low-affinity binding sites, respectively) remained unchanged after the treatment, as did the free-SH group requirements, storage stability and stereospecificity. Our results suggest that endogenous proteolytic activity could be involved in the increase of the number of membrane latent sites for L-triiodothyronine.

  20. Molecular Details of the PH Domain of ACAP1(BAR-PH) Protein Binding to PIP-Containing Membrane.

    PubMed

    Chan, Kevin Chun; Lu, Lanyuan; Sun, Fei; Fan, Jun

    2017-02-03

    ACAP1 proteins were previously reported to specifically bind PIP2-containing cell membranes and form well-structured protein lattices in order to conduct membrane tubulation. We carried out molecular dynamics simulations to characterize orientation of the PH domains with respect to the BAR domains inside the protein dimer. Followed by molecular dynamics simulations, we present a comprehensive orientation analysis of PH domain under different states including unbound and bound with lipids. We have examined two binding pockets on the PH domain and present PMF profiles of the two pockets to account for their preference to PIP2 lipids. Combining orientation analysis and studies of binding pockets, our simulations results reveal valuable molecular basis for protein-lipid interactions of ACAP1 proteins during membrane remodeling process.

  1. 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.

  2. 2,2'-Bis(monoacylglycero) PO4 (BMP), but Not 3,1'-BMP, Increases Membrane Curvature Stress to Enhance α-Tocopherol Transfer Protein Binding to Membranes

    SciTech Connect

    Baptist, Matilda; Panagabko, Candace; Nickels, Jonathan D.; Katsaras, John; Atkinson, Jeffrey

    2015-01-21

    Previous work revealed that α-tocopherol transfer protein (α-TTP) co-localizes with bis(monoacylglycero)phosphate (BMP) in late endosomes. BMP is a lipid unique to late endosomes and is believed to induce membrane curvature and support the multivesicular nature of this organelle. In this paper, we examined the effect of BMP on α-TTP binding to membranes using dual polarization interferometry and vesicle-binding assay. α-TTP binding to membranes is increased by the curvature-inducing lipid BMP. Finally, α-TTP binds to membranes with greater affinity when they contain the 2,2'-BMP versus 3,1'-BMP isomers.

  3. Nociceptin receptor binding in mouse forebrain membranes: thermodynamic characteristics and structure activity relationships

    PubMed Central

    Varani, K; Calo', G; Rizzi, A; Merighi, S; Toth, G; Guerrini, R; Salvadori, S; Borea, P A; Regoli, D

    1998-01-01

    The present study describes the labelling of the nociceptin (NC) receptor, ORL1, in mouse forebrain membranes with a new ligand partially protected from metabolic degradation at the C-terminal; the ligand, [3H]-NC-NH2, has a specific activity of 24.5 Ci mmol−1Saturation experiments revealed a single class of binding sites with a KD value of 0.55 nM and Bmax of 94 fmol mg−1 of protein. Non specific binding was 30% of total binding. Kinetic binding studies yielded the following rate constants: Kobs=0.104 min−1; K1=0.034 min−1; T1/2=20 min; K+1=0.07 min nM−1.Thermodynamic analyses indicated that [3H]-NC-NH2 binding to the mouse ORL1 is totally entropy driven, similar to what has been observed for the labelled agonists to the opioid receptors OP1(δ), OP2(κ) and OP3(μ).Receptor affinities of several NC fragments and analogues, including the newly discovered ORL-1 receptor antagonist [Phe1ψ(CH2-NH)Gly2]NC(1–13)-NH2 ([F/G]NC(1–13)-NH2), were also evaluated in displacement experiments. The competition curves for these compounds were found to be parallel to that of NC and the following order of potency was determined for NC fragments: NC-OH=NC-NH2=NC(1–13)-NH2 >> NC(1–12)-NH2 > NC(1–13)-OH >> NC(1–11)-NH2, and for NC and NC(1–13)-NH2 analogues: [Tyr1]NC-NH2 ⩾ [Leu1]NC(1–13)-NH2 ⩾ [Tyr1]NC(1–13)-NH2 ⩾ [F/G]NC(1–13)-NH2 >> [Phe3]NC(1–13)-NH2 > [DF/G]NC(1–13)-NH2.Standard opioid receptor ligands (either agonists or antagonists) were unable to displace [3H]-NC-NH2 binding when applied at concentrations up to 10 μM indicating that this new radioligand interacts with a non opioid site, probably the ORL1 receptor. PMID:9884077

  4. Highly potent antimicrobial peptides from N-terminal membrane-binding region of E. coli MreB

    PubMed Central

    Saikia, Karabi; Sravani, Yalavarthi Durga; Ramakrishnan, Vibin; Chaudhary, Nitin

    2017-01-01

    Microbial pathogenesis is a serious health concern. The threat escalates as the existing conventional antimicrobials are losing their efficacy against the evolving pathogens. Peptides hold promise to be developed into next-generation antibiotics. Antimicrobial peptides adopt amphipathic structures that could selectively bind to and disrupt the microbial membranes. Interaction of proteins with membranes is central to all living systems and we reasoned that the membrane-binding domains in microbial proteins could be developed into efficient antimicrobials. This is an interesting approach as self-like sequences could elude the microbial strategies of degrading the antimicrobial peptides, one of the mechanisms of showing resistance to antimicrobials. We selected the 9-residue-long membrane-binding region of E. coli MreB protein. The 9-residue peptide (C-terminal amide) and its N-terminal acetylated analog displayed broad-spectrum activity, killing Gram-negative bacteria, Gram-positive bacteria, and fungi. Extension with a tryptophan residue at the N-terminus drastically improved the activity of the peptides with lethal concentrations ≤10 μM against all the organisms tested. The tryptophan-extended peptides caused complete killing of C. albicans as well as gentamicin and methicillin resistant S. aureus at 5 μM concentration. Lipid-binding studies and electron microscopic analyses of the peptide-treated microbes suggest membrane disruption as the mechanism of killing. PMID:28230084

  5. Highly potent antimicrobial peptides from N-terminal membrane-binding region of E. coli MreB.

    PubMed

    Saikia, Karabi; Sravani, Yalavarthi Durga; Ramakrishnan, Vibin; Chaudhary, Nitin

    2017-02-23

    Microbial pathogenesis is a serious health concern. The threat escalates as the existing conventional antimicrobials are losing their efficacy against the evolving pathogens. Peptides hold promise to be developed into next-generation antibiotics. Antimicrobial peptides adopt amphipathic structures that could selectively bind to and disrupt the microbial membranes. Interaction of proteins with membranes is central to all living systems and we reasoned that the membrane-binding domains in microbial proteins could be developed into efficient antimicrobials. This is an interesting approach as self-like sequences could elude the microbial strategies of degrading the antimicrobial peptides, one of the mechanisms of showing resistance to antimicrobials. We selected the 9-residue-long membrane-binding region of E. coli MreB protein. The 9-residue peptide (C-terminal amide) and its N-terminal acetylated analog displayed broad-spectrum activity, killing Gram-negative bacteria, Gram-positive bacteria, and fungi. Extension with a tryptophan residue at the N-terminus drastically improved the activity of the peptides with lethal concentrations ≤10 μM against all the organisms tested. The tryptophan-extended peptides caused complete killing of C. albicans as well as gentamicin and methicillin resistant S. aureus at 5 μM concentration. Lipid-binding studies and electron microscopic analyses of the peptide-treated microbes suggest membrane disruption as the mechanism of killing.

  6. 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

  7. 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.

  8. Increased platelet membrane [3H]-LSD binding in patients on chronic neuroleptic treatment.

    PubMed Central

    Schächter, M; Geaney, D P; Grahame-Smith, D G; Cowen, P J; Elliott, J M

    1985-01-01

    Using a [3H]-lysergic acid diethylamide [( 3H]-LSD) binding technique, platelet 5-hydroxytryptamine (5-HT) receptor number and affinity were compared in schizophrenics treated with depot thioxanthenes and phenothiazines and controls. There was an approximately 30% increase in platelet receptor number (Bmax) in the patient group. There was a decrease in affinity (increase in Kd) of about 30% in the patient group. This was probably due to the persistence of the neuroleptic in the platelet membrane preparation. There was a weak positive correlation between receptor number and total neuroleptic dosage. The increased number of 5-HT receptors is consistent with the previously reported enhancement of 5-HT-induced platelet aggregation in patients treated with long-term phenothiazines and thioxanthenes. Our findings are compatible with 5-HT up-regulation in human platelets produced by depot neuroleptic therapy. It is not known whether parallel changes may be occurring in brain 5-HT receptors. PMID:2859873

  9. Identification of an Iron-Regulated, Hemin-Binding Outer Membrane Protein in Sinorhizobium meliloti

    PubMed Central

    Battistoni, Federico; Platero, Raúl; Duran, Rosario; Cerveñansky, Carlos; Battistoni, Julio; Arias, Alicia; Fabiano, Elena

    2002-01-01

    Rhizobia are soil bacteria that are able to establish symbiotic associations with leguminous hosts. In iron-limited environments these bacteria can use iron present in heme or heme compounds (hemoglobin, leghemoglobin). Here we report the presence in Sinorhizobium meliloti of an iron-regulated outer membrane protein that is able to bind hemin but not hemoglobin. Protein assignment was done by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Tryptic peptides correlated with the mass measurements obtained accounted for 54% of the translated sequence of a putative heme receptor gene present in the chromosome of S. meliloti 1021. The results which we obtained suggest that this protein (designated ShmR for Sinorhizobium heme receptor) is involved in high-affinity heme-mediated iron transport. PMID:12450806

  10. 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

  11. Plasmodium falciparum double C2 domain protein, PfDOC2, binds to calcium when associated with membranes.

    PubMed

    Jean, Sophonie; Zapata-Jenks, Mónica A; Farley, Julie M; Tracy, Erin; Mayer, D C Ghislaine

    2014-09-01

    The pathogenesis of malaria is strongly correlated with secretion of the micronemes, the apical organelles which contain the adhesins required for invasion of Plasmodium falciparum into human erythrocytes. A critical event in P. falciparum erythrocyte invasion is the production of calcium transients. After entering the cell, Ca(2+) binds to soluble Ca(2+)-binding proteins, such as the double C2 domains (DOC2). Recently, deletion of a P. falciparum DOC2 protein, PfDOC2, was shown to cause impairment in microneme secretion. However, PfDOC2 remains poorly characterized. Here, we report that PfDOC2 is expressed throughout the erythrocytic cycle and demonstrate that it is associated with membrane fractions and binds to calcium when it is part of these membranous structures. In summary, we show that PfDOC2 is a calcium lipid-binding protein of the protein kinase C type of DOC2 proteins.

  12. Presence of a low molecular weight endogenous inhibitor on 3H-muscimol binding in synaptic membranes

    NASA Astrophysics Data System (ADS)

    Yoneda, Yukio; Kuriyama, Kinya

    1980-06-01

    The specific binding of 3H-muscimol to synaptic membrane preparations obtained from the rat brain has been thought to reflect the association of γ-aminobutyric acid (GABA), a potential candidate as an inhibitory neurotransmitter in the mammalian central nervous system (CNS), with its synaptic receptors1,2. Treatment of synaptic membranes with Triton X-100 significantly increases the specific binding of 3H-muscimol2. Several reports also indicate the presence of endogenous substances, such as GABA3, acidic protein4 and phosphatidylethanolamine5, which inhibit Na-independent binding of 3H-GABA in the synaptic membranous fractions from the rat brain. We report here that in the supernatant obtained from Triton-treated synaptic membranes there exists a new type of endogenous inhibitor of 3H-muscimol binding which is apparently different from the inhibitory substances described previously3-5. The new inhibitor has a low molecular weight (MW) and probably originated from neurones rather than glial cells. We have termed this endogenous inhibitor the GABA receptor binding inhibitory factor (GRIF).

  13. Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase1

    PubMed Central

    Olivari, Claudio; Meanti, Cristina; De Michelis, Maria Ida; Rasi-Caldogno, Franca

    1998-01-01

    Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H+-ATPase in plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H+-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H+-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H+-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H+-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H+-ATPase antiserum and with an anti-14–3-3 antiserum indicated an FC-induced association of FCBP with the PM H+-ATPase. Analysis of the activation state of PM H+-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme. PMID:9489010

  14. How cholesterol interacts with membrane proteins: an exploration of cholesterol-binding sites including CRAC, CARC, and tilted domains.

    PubMed

    Fantini, Jacques; Barrantes, Francisco J

    2013-01-01

    The plasma membrane of eukaryotic cells contains several types of lipids displaying high biochemical variability in both their apolar moiety (e.g., the acyl chain of glycerolipids) and their polar head (e.g., the sugar structure of glycosphingolipids). Among these lipids, cholesterol is unique because its biochemical variability is almost exclusively restricted to the oxidation of its polar -OH group. Although generally considered the most rigid membrane lipid, cholesterol can adopt a broad range of conformations due to the flexibility of its isooctyl chain linked to the polycyclic sterane backbone. Moreover, cholesterol is an asymmetric molecule displaying a planar α face and a rough β face. Overall, these structural features open up a number of possible interactions between cholesterol and membrane lipids and proteins, consistent with the prominent regulatory functions that this unique lipid exerts on membrane components. The aim of this review is to describe how cholesterol interacts with membrane lipids and proteins at the molecular/atomic scale, with special emphasis on transmembrane domains of proteins containing either the consensus cholesterol-binding motifs CRAC and CARC or a tilted peptide. Despite their broad structural diversity, all these domains bind cholesterol through common molecular mechanisms, leading to the identification of a subset of amino acid residues that are overrepresented in both linear and three-dimensional membrane cholesterol-binding sites.

  15. Analysis of the induction of the myelin basic protein binding to the plasma membrane phospholipid monolayer

    NASA Astrophysics Data System (ADS)

    Zhang, Lei; Hao, Changchun; Feng, Ying; Gao, Feng; Lu, Xiaolong; Li, Junhua; Sun, Runguang

    2016-09-01

    Myelin basic protein (MBP) is an essential structure involved in the generation of central nervous system (CNS) myelin. Myelin shape has been described as liquid crystal structure of biological membrane. The interactions of MBP with monolayers of different lipid compositions are responsible for the multi-lamellar structure and stability of myelin. In this paper, we have designed MBP-incorporated model lipid monolayers and studied the phase behavior of MBP adsorbed on the plasma membrane at the air/water interface by thermodynamic method and atomic force microscopy (AFM). By analyzing the pressure-area (π-A) and pressure-time (π-T) isotherms, univariate linear regression equation was obtained. In addition, the elastic modulus, surface pressure increase, maximal insertion pressure, and synergy factor of monolayers were detected. These parameters can be used to modulate the monolayers binding of protein, and the results show that MBP has the strongest affinity for 1,2-dipalmitoyl-sn-glycero-3- phosphoserine (DPPS) monolayer, followed by DPPC/DPPS mixed and 1,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC) monolayers via electrostatic and hydrophobic interactions. AFM images of DPPS and DPPC/DPPS mixed monolayers in the presence of MBP (5 nM) show a phase separation texture at the surface pressure of 20 mN/m and the incorporation of MBP put into the DPPC monolayers has exerted a significant effect on the domain structure. MBP is not an integral membrane protein but, due to its positive charge, interacts with the lipid head groups and stabilizes the membranes. The interaction between MBP and phospholipid membrane to determine the nervous system of the disease has a good biophysical significance and medical value. Project supported by the National Natural Science Foundation of China (Grant Nos. 21402114 and 11544009), the Natural Science Basic Research Plan in Shaanxi Province of China (Grant No. 2016JM2010), the Fundamental Research Funds for the Central

  16. A comparison of insulin binding by liver plasma membranes of rats fed a high glucose diet or a high fat diet.

    PubMed

    Sun, J V; Tepperman, H M; Tepperman, J

    1977-07-01

    The interaction of (125)I-labeled insulin with purified liver plasma membrane from rats fed a high fat (L) diet or a high glucose (G) diet was studied with respect to specific binding, insulin degradation, binding site degradation, and rate of hormone association and dissociation. Scatchard analysis suggested the presence of high and low affinity binding sites for membranes of both G and L diet-adapted rats. However, liver plasma membrane from rats fed the high glucose diet bound 50% more insulin than did membrane from rats fed the high fat diet. Diet did not change insulin binding site degradation. The results suggested that an apparently reduced number of insulin binding sites (G = 10.2 +/- 2.45 x 10(-12) mol/mg membrane protein, L = 4.5 +/- 1.73 x 10(-12) mol/mg membrane protein) associated with fat feeding as compared to glucose feeding was responsible for the reduced insulin binding by membrane from rats fed the high fat diet. The effects of concanavalin A (Con A) on insulin binding to liver plasma membranes were also investigated. Con A enhanced the specific binding of insulin to liver plasma membranes from rats fed either diet at concentrations lower than 50 micro g/ml, whereas at concentrations higher than 50 micro g/ml Con A inhibited insulin binding to these membranes. The stimulatory effect of Con A on insulin binding at low concentrations was greater and inhibition of binding at high concentration was less in the case of membrane prepared from L diet-adapted animals. These results suggested that diet can modify the plasma membrane glycoproteins.

  17. Molecular binding of black tea theaflavins to biological membranes: relationship to bioactivities.

    PubMed

    Sirk, Timothy W; Friedman, Mendel; Brown, Eugene F

    2011-04-27

    Molecular dynamics simulations were used to study the interactions of three theaflavin compounds with lipid bilayers. Experimental studies have linked theaflavins to beneficial health effects, some of which are related to interactions with the cell membrane. The molecular interaction of theaflavins with membranes was explored by simulating the interactions of three theaflavin molecules (theaflavin, theaflavin-3-gallate, and theaflavin-3,3'-digallate) with a mixed bilayer composed of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE). The simulations show that the theaflavins evaluated have an affinity for the lipid bilayer surface via hydrogen bonding. The molecular structure of theaflavins influenced their configuration when binding to the bilayer surface, as well as their ability to form hydrogen bonds with the lipid headgroups. The theaflavin-bilayer interactions studied here help to define structure-function relationships of the theaflavins and provide a better understanding of the role of theaflavins in biological processes. The significance of the results are discussed in the context of black tea composition and bioactivity.

  18. 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

  19. Real-time analyses of retinol transport by the membrane receptor of plasma retinol binding protein.

    PubMed

    Kawaguchi, Riki; Zhong, Ming; Sun, Hui

    2013-01-28

    Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

  20. Magnetic bead isolation of neutrophil plasma membranes and quantification of membrane-associated guanine nucleotide binding proteins.

    PubMed

    Chang, Peter S; Absood, Afaf; Linderman, Jennifer J; Omann, Geneva M

    2004-02-15

    A protocol for isolation of neutrophil plasma membranes utilizing a plasma membrane marker antibody, anti-CD15, attached to superparamagnetic beads was developed. Cells were initially disrupted by nitrogen cavitation and then incubated with anti-CD15 antibody-conjugated superparamagnetic beads. The beads were then washed to remove unbound cellular debris and cytosol. Recovered plasma membranes were quantified by immunodetection of G(beta2) in Western blots. This membrane marker-based separation yielded highly pure plasma membranes. This protocol has advantages over standard density sedimentation protocols for isolating plasma membrane in that it is faster and easily accommodates cell numbers as low as 10(6). These methods were coupled with immunodetection methods and an adenosine 5(')-diphosphate-ribosylation assay to measure the amount of membrane-associated G(ialpha) proteins available for receptor coupling in neutrophils either stimulated with N-formyl peptides or treated to differing degrees with pertussis toxin. As expected, pertussis toxin treatment decreased the amount of membrane G protein available for signaling although total membrane G protein was not affected. In addition, activation of neutrophils with N-formyl peptides resulted in an approximately 50% decrease in G protein associated with the plasma membrane.

  1. Despite a Conserved Cystine Knot Motif, Different Cyclotides Have Different Membrane Binding Modes

    PubMed Central

    Wang, Conan K.; Colgrave, Michelle L.; Ireland, David C.; Kaas, Quentin; Craik, David J.

    2009-01-01

    Abstract Cyclotides are cyclic proteins produced by plants for defense against pests. Because of their remarkable stability and diverse bioactivities, they have a range of potential therapeutic applications. The bioactivities of cyclotides are believed to be mediated through membrane interactions. To determine the structural basis for the biological activity of the two major subfamilies of cyclotides, we determined the conformation and orientation of kalata B2 (kB2), a Möbius cyclotide, and cycloviolacin O2 (cO2), a bracelet cyclotide, bound to dodecylphosphocholine micelles, using NMR spectroscopy in the presence and absence of 5- and 16-doxylstearate relaxation probes. Analysis of binding curves using the Langmuir isotherm indicated that cO2 and kB2 have association constants of 7.0 × 103 M−1 and 6.0 × 103 M−1, respectively, consistent with the notion that they are bound near the surface, rather than buried deeply within the micelle. This suggestion is supported by the selective broadening of micelle-bound cyclotide NMR signals upon addition of paramagnetic Mn ions. The cyclotides from the different subfamilies exhibited clearly different binding orientations at the micelle surface. Structural analysis of cO2 confirmed that the main element of the secondary structure is a β-hairpin centered in loop 5. A small helical turn is present in loop 3. Analysis of the surface profile of cO2 shows that a hydrophobic patch stretches over loops 2 and 3, in contrast to the hydrophobic patch of kB2, which predominantly involves loops 2 and 5. The different location of the hydrophobic patches in the two cyclotides explains their different binding orientations and provides an insight into the biological activities of cyclotides. PMID:19720036

  2. Components of the plasma membrane of growing axons. III. Saxitoxin binding to sodium channels

    PubMed Central

    1984-01-01

    The density of sodium channels was measured in growing and mature axons of the olfactory nerve of the bullfrog, using as a probe the drug saxitoxin (STX). The toxin binds to control nerves from adult animals in a saturable manner with a dissociation constant of approximately 23 nM at 4 degrees C and a capacity of 72 fmol/mg wet weight, equivalent to about five sites per square micrometer of axolemma. In growing nerves, obtained from adult frogs 4-5 wk following removal of the original nerve, the STX-binding capacity per wet weight of tissue is markedly reduced, to approximately 25% of control values, and appears to decrease in the proximodistal direction. STX-binding data, expressed as STX/mg wet weight, was converted to STX/micron 2 of axolemma using stereologically derived values of membrane area per milligram wet weight of nerve. The axolemmal content (area/mg wet weight) of all regions of growing nerve is substantially decreased compared to controls, but increases in the proximodistal direction by 60%. These changes in axolemmal area result in calculated STX receptor densities (per unit axolemmal area) which, in distal regions, are approximately at the level of the mature nerve and, in proximal regions, are actually increased above controls by 50 to 70%. Upon comparing the axolemmal density of intramembrane particles, reported in the companion paper, with the calculated density of STX receptors in both mature and growing nerves, we find a correlation between STX receptors and intramembrane particles with diameters of 11.5-14.0 nm. The growing axon's gradient of sodium channels and the shift from this gradient to a uniform distribution in the mature axon suggest (a) that sodium channels are inserted into the perikaryal plasmalemma and diffuse from there into the growing axolemma, and (b) that the axolemma undergoes functional maturation during growth. PMID:6325471

  3. Binding equilibrium and kinetics of membrane-anchored receptors and ligands in cell adhesion: Insights from computational model systems and theory.

    PubMed

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

    2016-09-02

    The adhesion of cell membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. In this article, we review recent results from simulations and theory that lead to novel insights on how the binding equilibrium and kinetics of these proteins is affected by the membranes and by the membrane anchoring and molecular properties of the proteins. Simulations and theory both indicate that the binding equilibrium constant [Formula: see text] and the on- and off-rate constants of anchored receptors and ligands in their 2-dimensional (2D) membrane environment strongly depend on the membrane roughness from thermally excited shape fluctuations on nanoscales. Recent theory corroborated by simulations provides a general relation between [Formula: see text] and the binding constant [Formula: see text] of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in 3 dimensions (3D).

  4. 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.

  5. Escherichia coli phage-shock protein A (PspA) binds to membrane phospholipids and repairs proton leakage of the damaged membranes.

    PubMed

    Kobayashi, Ryuji; Suzuki, Toshiharu; Yoshida, Masasuke

    2007-10-01

    Escherichia coli phage-shock protein A (PspA), a 25.3 kDa peripheral membrane protein, is induced under the membrane stress conditions and is assumed to help maintain membrane potential. Here, we report that purified PspA, existing as a large oligomer, is really able to suppress proton leakage of the membranes. This was demonstrated for membrane vesicles prepared from the PspA-lacking E. coli mutants, and for membrane vesicles damaged by ethanol and Triton X-100 prepared from the mutant and the wild-type cells. PspA also suppressed proton leakage of damaged liposomes made from E. coli total lipids. Furthermore, we found that PspA bound preferentially to liposomes containing phosphatidylserine and phosphatidylglycerol. All these effects were not observed for monomer PspA that was prepared by refolding of urea-denatured PspA. These results indicate that oligomers of PspA bind to membrane phospholipids and suppress proton leakage.

  6. Androgens and women's health.

    PubMed

    Redmond, G P

    1998-01-01

    Androgenic disorders are those conditions in women characterized by excessive androgen action. They are the most common endocrinopathy of women, affecting from 10% to 20%. Signs are: persistent acne, hirsutism and androgenic alopecia, which is the female equivalent of male pattern baldness. A subgroup, those traditionally labeled as having polycystic ovary syndrome (PCOS), additionally have anovulation, as well as menstrual abnormalities and, often, obesity. Although women with androgenic disorders usually present themselves for help with the skin or menstrual changes, there are other important implications regarding their health. Women with PCOS have varying degrees of insulin resistance, and an increased incidence of Type II diabetes mellitus, as well as unfavorable lipid patterns. The presence of these risk factors is suggested by upper segment obesity, darkening of the skin, and the other skin changes that make up acanthosis nigricans. Diagnosis involves measurement of circulating androgens (of which free testosterone is most important), together with prolactin and FSH when menstrual dysfunction is present. Many women with androgenic skin changes have normal serum androgen levels, suggesting increased end organ sensitivity to androgens. Others have hyperandrogenism (of ovarian or adrenal origin). Treatment is usually successful in controlling acne, reducing hirsutism and stabilizing, or partially reversing, androgenic alopecia. Pharmacological approaches involve suppressing androgen levels, for example, the use of an appropriate oral contraceptive, or antagonizing androgen action with several medications that have this activity. Unfortunately, most women with androgenic disorders are frustrated in their efforts to obtain medical help. Understanding androgenic disorders will enable the physician to significantly help the majority of women with these conditions.

  7. 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.).

  8. 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.

  9. Mechanism and functions of membrane binding by the Atg5-Atg12/Atg16 complex during autophagosome formation.

    PubMed

    Romanov, Julia; Walczak, Marta; Ibiricu, Iosune; Schüchner, Stefan; Ogris, Egon; Kraft, Claudine; Martens, Sascha

    2012-11-14

    Autophagy is a conserved process for the bulk degradation of cytoplasmic material. Triggering of autophagy results in the formation of double membrane-bound vesicles termed autophagosomes. The conserved Atg5-Atg12/Atg16 complex is essential for autophagosome formation. Here, we show that the yeast Atg5-Atg12/Atg16 complex directly binds membranes. Membrane binding is mediated by Atg5, inhibited by Atg12 and activated by Atg16. In a fully reconstituted system using giant unilamellar vesicles and recombinant proteins, we reveal that all components of the complex are required for efficient promotion of Atg8 conjugation to phosphatidylethanolamine and are able to assign precise functions to all of its components during this process. In addition, we report that in vitro the Atg5-Atg12/Atg16 complex is able to tether membranes independently of Atg8. Furthermore, we show that membrane binding by Atg5 is downstream of its recruitment to the pre-autophagosomal structure but is essential for autophagy and cytoplasm-to-vacuole transport at a stage preceding Atg8 conjugation and vesicle closure. Our findings provide important insights into the mechanism of action of the Atg5-Atg12/Atg16 complex during autophagosome formation.

  10. Membrane binding properties of EBV gp110 C-terminal domain; evidences for structural transition in the membrane environment

    SciTech Connect

    Park, Sung Jean; Seo, Min-Duk; Lee, Suk Kyeong; Lee, Bong Jin

    2008-09-30

    Gp110 of Epstein-Barr virus (EBV) mainly localizes on nuclear/ER membranes and plays a role in the assembly of EBV nucleocapsid. The C-terminal tail domain (gp110 CTD) is essential for the function of gp110 and the nuclear/ER membranes localization of gp110 is ruled by its C-terminal unique nuclear localization signal (NLS), consecutive four arginines. In the present study, the structural properties of gp110 CTD in membrane mimics were investigated using CD, size-exclusion chromatography, and NMR, to elucidate the effect of membrane environment on the structural transition and to compare the structural feature of the protein in the solution state with that of the membrane-bound form. CD and NMR analysis showed that gp110 CTD in a buffer solution appears to adopt a stable folding intermediate which lacks compactness, and a highly helical structure is formed only in membrane environments. The helical content of gp110 CTD was significantly affected by the negative charge as well as the size of membrane mimics. Based on the elution profiles of the size-exclusion chromatography, we found that gp110 CTD intrinsically forms a trimer, revealing that a trimerization region may exist in the C-terminal domain of gp110 like the ectodomain of gp110. The mutation of NLS (RRRR) to RTTR does not affect the overall structure of gp110 CTD in membrane mimics, while the helical propensity in a buffer solution was slightly different between the wild-type and the mutant proteins. This result suggests that not only the helicity induced in membrane environment but also the local structure around NLS may be related to trafficking to the nuclear membrane. More detailed structural difference between the wild-type and the mutant in membrane environment was examined using synthetic two peptides including the wild-type NLS and the mutant NLS.

  11. Mutagenesis of Paramyxovirus Hemagglutinin-Neuraminidase Membrane-Proximal Stalk Region Influences Stability, Receptor Binding, and Neuraminidase Activity

    PubMed Central

    Adu-Gyamfi, Emmanuel; Kim, Lori S.; Jardetzky, Theodore S.

    2016-01-01

    ABSTRACT Paramyxoviridae consist of a large family of enveloped, negative-sense, nonsegmented single-stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN [also called H or G depending on virus type and substrate]), which acts as a receptor binding protein, and a fusion (F) protein, which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN 4-helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross-linking as well as functional assays to investigate the role of the structurally unresolved membrane-proximal stalk region (MPSR) (residues 37 to 58) of HN in the context of headless and full-length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F triggering. IMPORTANCE Paramyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F), to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane-anchored flexible stalk of ∼80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane-proximal stalk region (MPSR) (HN, residues 37 to 56) of the paramyxovirus parainfluenza virus

  12. Hybrid In Silico/In Vitro Approaches for the Identification of Functional Cholesterol-Binding Domains in Membrane Proteins.

    PubMed

    Di Scala, Coralie; Fantini, Jacques

    2017-01-01

    In eukaryotic cells, cholesterol is an important regulator of a broad range of membrane proteins, including receptors, transporters, and ion channels. Understanding how cholesterol interacts with membrane proteins is a difficult task because structural data of these proteins complexed with cholesterol are scarce. Here, we describe a dual approach based on in silico studies of protein-cholesterol interactions, combined with physico-chemical measurements of protein insertion into cholesterol-containing monolayers. Our algorithm is validated through careful analysis of the effect of key mutations within and outside the predicted cholesterol-binding site. Our method is illustrated by a complete analysis of cholesterol-binding to Alzheimer's β-amyloid peptide, a protein that penetrates the plasma membrane of brain cells through a cholesterol-dependent process.

  13. Kinetics of membrane binding and dissociation of 5-fluorouracil by pulsed-field-gradient 19F NMR

    NASA Astrophysics Data System (ADS)

    Yoshii, Noriyuki; Okamura, Emiko

    2009-06-01

    The kinetics of membrane binding and dissociation of an anticancer drug, 5-fluorouracil (5FU) is quantified by high resolution NMR with the pulsed-field-gradient technique. The 19F NMR signal of 5FU is analyzed at 293-313 K by the solution of Bloch equation with exchange terms. The rate constants of 5FU binding and dissociation are 0.2 and 4.1 s -1 at 303 K. The 5FU motion in the vertical direction to the membrane surface is restricted as compared with the lateral diffusion, judging from the activation energy (57 kJ/mol) larger than the lateral diffusion in membrane (26 kJ/mol [E. Okamura, N. Yoshii, J. Chem. Phys. 129 (2008) 215102]).

  14. Characterization of the binding of [3H]-SB-204269, a radiolabelled form of the new anticonvulsant SB-204269, to a novel binding site in rat brain membranes

    PubMed Central

    Herdon, Hugh J; Jerman, Jeffrey C; Stean, Tania O; Middlemiss, Derek N; Chan, Wai N; Vong, Antonio K; Evans, John M; Thompson, Mervyn; Upton, Neil

    1997-01-01

    SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-benzol[b]pyran-3R-ol, hemihydrate) shows potent anticonvulsant activity in a range of animal seizure models, with a lack of neurological or cardiovascular side-effects. The profile of the compound suggests that it may have a novel mechanism of action. This study describes the characteristics of a binding site for [3H]-SB-204269 in rat forebrain membranes. Specific [3H]-SB-204269 binding was saturable and analysis indicated binding to a homogenoeous population of non-interacting binding sites with a dissociation constant (KD) of 32±1 nM and a maximum binding capacity (Bmax) of 253±18 fmol mg−1 protein. Kinetic studies indicated monophasic association and dissociation. Binding was similar in HEPES or Tris-HCl buffers and was unaffected by Na+, K+, Ca2+ or Mg2+ ions. Specific binding was widely distributed in brain, but was minimal in a range of peripheral tissues. Specific [3H]-SB-204269 binding was highly stereoselective, with a 1000 fold difference between the affinities of SB-204269 and its enantiomer SB-204268 for the binding site. The affinities of analogues of SB-204269 for binding can be related to their activities in the mouse maximal electroshock seizure threshold (MEST) test of anticonvulsant action. None of the standard anticonvulsant drugs, phenobarbitone, phenytoin, sodium valproate, carbamazepine, diazepam and ethosuximide, or the newer anticonvulsants, lamotrigine, vigabatrin, gabapentin and levetiracetam, showed any affinity for the [3H]-SB-204269 binding site. A wide range of drugs active at amino acid receptors, Na+ or K+ channels or various other receptors did not demonstrate any affinity for the binding site. These studies indicate that SB-204269 possesses a specific CNS binding site which may mediate its anticonvulsant activity. This binding site does not appear to be directly related to the sites of action of other known anticonvulsant agents, but may

  15. Comparison of binding parameters of sigma 1 and sigma 2 binding sites in rat and guinea pig brain membranes: novel subtype-selective trishomocubanes.

    PubMed

    Nguyen, V H; Kassiou, M; Johnston, G A; Christie, M J

    1996-09-12

    Comparisons of binding parameters of [3H](+)-pentazocine and [3H]1,3-di-o-tolylguanidine (DTG) at sigma binding sites in guinea pig and rat brain membranes demonstrated that [3H](+)-pentazocine binds to a single high-affinity site, whereas [3H]DTG binds to two high-affinity sites in both species. The Kd values of the radioligands were similar in both types of membranes. However, the density of sigma 1 sites in guinea pig was significantly higher than that of rat. Novel trishomocubanes were tested for their affinities at sigma 1 and sigma 2 binding sites in guinea pig brain membranes using [3H](+)-pentazocine and [3H]DTG as the radioligands. N-(4-Phenylbutyl)-3-hydroxy-4- azahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11)]dodecane (ANSTO-14) showed the highest affinity for the sigma 1 site (Ki = 9.4 nM) and 19-fold sigma 1/sigma 2 selectivity, as a result of increasing the alkyl chain between the cubane moiety and the aromatic ring. N-(3'-Fluorophenyl)methyl- 3-hydroxy-4-azahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11]dodeca ne (ANSTO-19), displayed the highest affinity for sigma 2 sites (Ki = 19.6 nM) and 8-fold sigma 2/sigma 1 selectivity due to a fluoro substitution in the meta position of the aromatic ring. These represent structurally novel lead compounds, especially for the development of selective sigma 2 receptor ligands.

  16. Cytochalasin B binding proteins in human erythrocyte membranes. Modulation of glucose sensitivity by site interaction and partial solubilization of binding activities.

    PubMed

    Pinkofsky, H B; Rampal, A L; Cowden, M A; Jung, C Y

    1978-07-25

    We have previously described three different cytochalasin B binding sites in human erythrocyte membranes, a D-glucose-sensitive site (Site I), a cytochalasin E-sensitive site (Site II), and a site (Site III) insensitive to both D-glucose and cytochalasin E. Ligand bindings to each of these sites were considered to be independent (Jung, C., and Rampal, A. (1977) J. Biol. Chem. 252, 5456-5463). However, we have obtained subsequently the following evidence which indicated that an interaction occurs between Sites II and III, and this modulates sensitivity of Site III to the sugar. The displacement of cytochalasin E greatly exceeds the sum of their independent displacements. This ghosts extracted with EDTA or 2,3-dimethylmaleic anhydride at low ionic strength lack Site II activity but retain Site I and III activities, and both of these activities are displaceable by D-glucose alone. This indicated that the removal of Site II from the membrane confers glucose sensitivity to Site III. These observations are consistent with a model that Sites II and III in the membrane exist in a close association through which unliganded Site II maintains the glucose insensitivity of Site III, and once site II is liganded or removed by extraction this association is disrupted and Site III becomes glucose-sensitive. The ghosts extracted with Triton X-100 retain a cytochalasin B binding activity similar to that of site II (Kd = 1.8 X 10(-7) M, cytochalasin E-sensitive, glucose-insensitive), whereas a binding activity similar to that of Site I (Kd = 4 X 10(-7) M, cytochalasin E-insensitive, glucose-sensitive) is recovered in the Triton extract. A cytochalasin B binding activity similar to that of Site II is solubilized by EDTA at low ionic strength.

  17. Specificity of Auxin-binding Sites on Maize Coleoptile Membranes as Possible Receptor Sites for Auxin Action 1

    PubMed Central

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

    1977-01-01

    Dissociation coefficients of auxin-binding sites on maize (Zea mays L.) coleoptile membranes were measured, for 48 auxins and related ring compounds, by competitive displacement of 14C-naphthaleneacetic acid from the binding sites. The sites bind with high affinity several ring compounds with acidic side chains 2 to 4 carbons long, and much more weakly bind neutral ring compounds and phenols related to these active acids, most phenoxyalkylcarboxylic acids, and arylcarboxylic acids except benzoic acid, which scarcely binds, and triiodobenzoic acids, which bind strongly. Specificity of the binding is narrowed in the presence of a low molecular weight “supernatant factor” that occurs in maize and other tissues. Activity of many of the analogs as auxin agonists or antagonists in the cell elongation response was determined with maize coleoptiles. These activities on the whole roughly parallel the affinities of the binding sites for the same compounds, especially affinities measured in the presence of supernatant factor, but there are some quantitative discrepancies, especially among phenoxyalkylcarboxylic acids. In view of several factors that can cause receptor affinity and biological activity values to diverge quantitatively among analogs, the findings appear to support the presumption that the auxin-binding sites may be receptors for auxin action. PMID:16660143

  18. Oncogenic K-Ras Binds to an Anionic Membrane in Two Distinct Orientations: A Molecular Dynamics Analysis

    PubMed Central

    Prakash, Priyanka; Zhou, Yong; Liang, Hong; Hancock, John F.; Gorfe, Alemayehu A.

    2016-01-01

    K-Ras is a membrane-associated GTPase that cycles between active and inactive conformational states to regulate a variety of cell signaling pathways. Somatic mutations in K-Ras are linked to 15–20% of all human tumors. K-Ras attaches to the inner leaflet of the plasma membrane via a farnesylated polybasic domain; however, the structural details of the complex remain poorly understood. Based on extensive (7.5 μs total) atomistic molecular dynamics simulations here we show that oncogenic mutant K-Ras interacts with a negatively charged lipid bilayer membrane in multiple orientations. Of these, two highly populated orientations account for ∼54% of the conformers whose catalytic domain directly interacts with the bilayer. In one of these orientation states, membrane binding involves helices 3 and 4 of the catalytic domain in addition to the farnesyl and polybasic motifs. In the other orientation, β-strands 1–3 and helix 2 on the opposite face of the catalytic domain contribute to membrane binding. Flexibility of the linker region was found to be important for the reorientation. The biological significance of these observations was evaluated by initial experiments in cells overexpressing mutant K-Ras as well as by an analysis of Ras-effector complex structures. The results suggest that only one of the two major orientation states is capable of effector binding. We propose that the different modes of membrane binding may be exploited in structure-based drug design efforts for cancer therapy. PMID:26958889

  19. The non-specific membrane binding properties of delta9-tetrahydrocannabinol and the effects of various solubilizers.

    PubMed

    Roth, S H; Williams, P J

    1979-04-01

    The binding of [3H]delta9-tetrahydrocannabinol to crude and purified synaptosomal membrane suspended in either Krebs solution or 10 mM sodium phosphate buffer was examined. The membrane/buffer partition coefficient was found to be 12,500, and was constant over a free concentration range of 10(-8) to 10(-6)M. Binding was similar in both suspending media, and to both crude and purified synaptosomal membrane. The solubilizing agent Cremophor E.L. (8 microgram ml-1) decreased the partition coefficient by one-half, and by greater than 99% at 0.4 mg ml-1. Similar effects were observed with Tween 80, while ethanol caused a maximum decrease of 60%. Membrane concentrations of THC were calculated at various effective concentrations reported in the literature, and were within the range predicted by the Meyer-Overton rule of anaesthesia. An apparent non-specific interaction with neuronal membranes and effective membrane concentrations of the order 2 x 10(-4) to 1 x 10(-2) mol kg-1 suggests THC may exert some of its effects by a mechanism analogous to the general anaesthetics, and thus may be classified as a partial anaesthetic.

  20. Immune labeling and purification of a 71-kDa glutamate-binding protein from brain synaptic membranes

    SciTech Connect

    Chen, J.W.; Cunningham, M.D.; Galton, N.; Michaelis, E.K.

    1988-01-05

    Immunoblot studies of synaptic membranes isolated from rat brain using antibodies raised against a previously purified glutamate-binding protein (GBP) indicated labeling of an approx. 70-kDa protein band. Since the antibodies used were raised against a 14-kDa GBP, the present studies were undertaken to explore the possibility that the 14-kDa protein may have been a proteolytic fragment of a larger M/sub r/ protein in synaptic membranes. The major protein enriched in the most highly purified fractions was a 71-kDa glycoprotein, but a 63-kDa protein was co-purified during most steps of the isolation procedure. The glutamate-binding characteristics of these isolated protein fractions were very similar to those previously described for the 14-kDa GBP, including estimated dissociation constants for L-glutamate binding of 0.25 and 1 /sup +/M, inhibition of glutamate binding by azide and cyanide, and a selectivity of the ligand binding site for L-glutamate and L-aspartate. The neuroexcitatory analogs of L-glutamate and L-aspartate, ibotenate, quisqualate, and D-glutamate, inhibited L(/sup 3/H)glutamate binding to the isolated proteins, as did the antagonist of L-glutamate-induced neuronal excitation, L-glutamate diethylester. On the basis of the lack of any detectable glutamate-related enzyme activity associated with the isolated proteins and the presence of distinguishing sensitivities to analogs that inhibit glutamate transport carriers in synaptic membranes, it is proposed that the 71-kDa protein may be a component of a physiologic glutamate receptor complex in neuronal membranes.

  1. Adolescent androgenic alopecia.

    PubMed

    McDonough, Patrick Henry; Schwartz, Robert A

    2011-10-01

    Adolescent androgenic alopecia is pattern hair loss occurring in boys and girls younger than 18 years, whereas early-onset androgenic alopecia refers to pattern hair loss before 35 years of age. A number of studies published in the last decade have helped to elucidate the prevalence of adolescent androgenic alopecia, have clarified the genetic as well as physiologic mechanisms underlying hair loss, and have revealed the associated psychologic and systemic morbidities. This article provides an overview of the pathophysiology, diagnosis, and treatment of adolescent androgenic alopecia.

  2. Interactions between Plasmodium falciparum skeleton-binding protein 1 and the membrane skeleton of malaria-infected red blood cells.

    PubMed

    Kats, Lev M; Proellocks, Nicholas I; 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

    2015-07-01

    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 sub-domains 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.

  3. Nuclear exclusion of the androgen receptor by melatonin.

    PubMed

    Rimler, Avi; Culig, Zoran; Lupowitz, Zippora; Zisapel, Nava

    2002-05-01

    Androgen receptors (AR) play a crucial role in androgen-mediated processes and prostate cancer progression. The pineal hormone melatonin attenuates the androgen-dependent growth of benign and cancer prostate epithelial cells in vitro and may reverse clinical resistance to androgen ablation therapy in patients progressing on gonadotropin releasing hormone (GnRH) analogue. Where along the AR cascade does melatonin act remains to be determined. The effects of melatonin on AR localization, level and activity were assessed using androgen-insensitive prostate carcinoma PC3 cells stably transfected with a wild-type AR-expressing vector (PC3-AR).AR was localized to the PC3-AR cell nucleus in the absence of dihydrotestosterone (DHT). Melatonin caused a robust exclusion of the AR from the cell nucleus to the cytoplasm. The nuclear export inhibitor, leptomycin B prevented this process. The exclusion was selective since melatonin had no such effect on the nuclear localization of estrogen receptors alpha (ERalpha) in these cells. Melatonin also caused nuclear exclusion of the AR in the presence of DHT. In addition, it attenuated androgen induced reporter gene activity in PC3 cells co-transfected with the human AR and AR reporter plasmids. Elevated androgen concentrations counteracted melatonin's effects. Melatonin did not decrease AR level or androgen binding in the cells. The nuclear localization of the AR is a hallmark of its cellular activity. These data point to AR nuclear exclusion as a possible mechanism to attenuate androgen responses in target tissues.

  4. Enhanced membrane binding of autoantibodies to cultured keratinocytes of systemic lupus erythematosus patients after ultraviolet B/ultraviolet A irradiation.

    PubMed Central

    Golan, T D; Elkon, K B; Gharavi, A E; Krueger, J G

    1992-01-01

    Although sunlight is known to induce skin lesions in patients with systemic lupus erythematosus (SLE) and to exacerbate systemic manifestations, the underlying mechanisms remain obscure. We report experiments that show enhanced binding of IgG autoantibodies to the cell surface membrane of ultraviolet-B (UVB) irradiated (200-1,600 J/m2) cultured SLE keratinocytes in 10 out of 12 such cell strains. The autoantibody probes showing increased binding were directed against the soluble intracellular antigens, Sm, RNP, SSA/Ro, SSB/La, whereas serum with anti-dsDNA activity did not demonstrate such binding. Control keratinocytes from several sources shared low level binding of autoantibodies after ultraviolet light exposure. In addition, 4/6 UVB-sensitive SLE strains showed increased autoantibody binding to the surface of SLE keratinocytes after UVA exposure (50-150 kJ/m2), but of lower magnitude. When UVB-sensitive nonirradiated SLE strains were exposed to autologous serum, 3/8 sera demonstrated a striking increase in IgG binding, which increased further after UVB exposure. Enhanced expression of saline-soluble intracellular antigens on the cell surface membrane of patient, but not control, keratinocytes may, in part, explain the photosensitivity of patients with SLE. Images PMID:1522215

  5. Development of a Machine Learning Method to Predict Membrane Protein-Ligand Binding Residues Using Basic Sequence Information

    PubMed Central

    Suresh, M. Xavier; Gromiha, M. Michael; Suwa, Makiko

    2015-01-01

    Locating ligand binding sites and finding the functionally important residues from protein sequences as well as structures became one of the challenges in understanding their function. Hence a Naïve Bayes classifier has been trained to predict whether a given amino acid residue in membrane protein sequence is a ligand binding residue or not using only sequence based information. The input to the classifier consists of the features of the target residue and two sequence neighbors on each side of the target residue. The classifier is trained and evaluated on a nonredundant set of 42 sequences (chains with at least one transmembrane domain) from 31 alpha-helical membrane proteins. The classifier achieves an overall accuracy of 70.7% with 72.5% specificity and 61.1% sensitivity in identifying ligand binding residues from sequence. The classifier performs better when the sequence is encoded by psi-blast generated PSSM profiles. Assessment of the predictions in the context of three-dimensional structures of proteins reveals the effectiveness of this method in identifying ligand binding sites from sequence information. In 83.3% (35 out of 42) of the proteins, the classifier identifies the ligand binding sites by correctly recognizing more than half of the binding residues. This will be useful to protein engineers in exploiting potential residues for functional assessment. PMID:25802517

  6. 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.

  7. Brush border membrane binding properties of Bacillus thuringiensis Vip3A toxin to Heliothis virescens and Helicoverpa zea midguts.

    PubMed

    Lee, Mi Kyong; Miles, Paul; Chen, Jeng-Shong

    2006-01-27

    The binding properties of Vip3A, a new family of Bacillus thuringiensis insecticidal toxins, have been examined in the major cotton pests, Heliothis virescens and Helicoverpa zea. Vip3A bound specifically to brush border membrane vesicles (BBMV) prepared from both insect larval midguts. In order to examine the cross-resistance potential of Vip3A to the commercially available Cry1Ac and Cry2Ab2 toxins, the membrane binding site relationship among these toxins was investigated. Competition binding assays demonstrated that Vip3A does not inhibit the binding of either Cry1Ac or Cry2Ab2 and vice versa. BBMV protein blotting experiments showed that Vip3A does not bind to the known Cry1Ac receptors. These distinct binding properties and the unique protein sequence of Vip3A support its use as a novel insecticidal agent. This study indicates a very low cross-resistance potential between Vip3A and currently deployed Cry toxins and hence supports its use in an effective resistance management strategy in cotton.

  8. A novel point mutation (R840S) in the androgen receptor in a Brazilian family with partial androgen insensitivity syndrome.

    PubMed

    Melo, K F; Latronico, A C; Costa, E M; Billerbeck, A E; Mendonca, B B; Arnhold, I J

    1999-10-01

    Mutations of the androgen receptor gene causing androgen insensitivity syndrome in 46, XY individuals, result in phenotypes ranging from complete female to ambiguous genitalia to males with minor degrees of undervirilization. We studied two Brazilian brothers with partial androgen insensitivity syndrome. They were born with perineal hypospadias, bifid scrotum, small penis and cryptorchidism, and developed gynecomastia at puberty. Genomic DNA was extracted and denaturinggradient gel electrophoresis of exon 7 of the androgen receptor gene followed by sequence analysis revealed a new mutation, a C A transversion, altering codon 840 from arginine (CGT) to serine (AGT). R840 is located in the androgen binding domain, in a "hot spot" region, important for the formation and function of the hormone receptor-complex and within the region that is involved in androgen receptor dimerization. Replacement of arginine (basic) by serine (neutral and polar) is a nonconservative substitution. Three mutations in this residue (R840C, R840G nonconservative and R840H, conservative) were previously reported in patients with partial androgen insensitivity syndrome and when expressed "in vitro" lead to a subnormal transactivation of a reporter gene. We conclude that the novel R840 mutation in the androgen receptor is the cause of partial androgen insensitivity syndrome in this Brazilian family.

  9. 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

  10. Spectroscopic investigations of the binding mechanisms between antimicrobial peptides and membrane models of Pseudomonas aeruginosa and Klebsiella pneumoniae.

    PubMed

    Chai, Hanbo; Allen, William E; Hicks, Rickey P

    2014-08-01

    CD spectroscopy was used to investigate the interactions of a series of synthetic AMPs with LPS isolated from Pseudomonas aeruginosa and Klebsiella pneumoniae, as well as with various phospholipids to better approximate the chemical composition of the membranes of these two strains of Gram-negative bacteria. This investigation was conducted in order to probe how the contributions of key physicochemical properties of an AMP vary in different regions of the membranes of these two bacteria. The conclusions from this study are as follows. (1) The binding interactions between the AMP and the membranes are defined by the complementarity of delocalization of positive charge density of the basic amino side chains (i.e., electrostatics), molecular flexibility of the peptide backbone, and overall hydrophobicity. (2) The binding interactions of these AMPs to LPS seem to be predominantly with the lipid A region of the LPS. (3) Incorporation of phospholipids into the LPS containing SUVs resulted in dramatic changes in the conformational equilibrium of the bound AMPs. (4) For the LPS-phospholipid models of Pseudomonas aeruginosa, delocalization of the side chain positive charge plays a major role in determining the number of conformers that contribute to the binding conformational equilibrium. This relationship was not observed for the models of the outer and inner membranes of Klebsiella pneumoniae.

  11. 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

  12. 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.

  13. 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

  14. The novel antiepileptic drug levetiracetam (ucb L059) appears to act via a specific binding site in CNS membranes.

    PubMed

    Noyer, M; Gillard, M; Matagne, A; Hénichart, J P; Wülfert, E

    1995-11-14

    Levetiracetam ((S)-alpha-ethyl-2-oxo-pyrrolidine acetamide, ucb L059) is a novel potential antiepileptic agent presently in clinical development with unknown mechanism of action. The finding that its anticonvulsant activity is highly stereoselective (Gower et al., 1992) led us to investigate the presence of specific binding sites for [3H]levetiracetam in rat central nervous system (CNS). Binding assays, performed on crude membranes, revealed the existence of a reversible, saturable and stereoselective specific binding site. Results obtained in hippocampal membranes suggest that [3H]levetiracetam labels a single class of binding sites (nH = 0.92 +/- 0.06) with modest affinity (Kd = 780 +/- 115 nM) and with a high binding capacity (Bmax = 9.1 +/- 1.2 pmol/mg protein). Similar Kd and Bmax values were obtained in other brain regions (cortex, cerebellum and striatum). ucb L060, the (R) enantiomer of levetiracetam, displayed about 1000 times less affinity for these sites. The binding of [3H]levetiracetam is confined to the synaptic plasma membranes in the central nervous system since no specific binding was observed in a range of peripheral tissues including heart, kidneys, spleen, pancreas, adrenals, lungs and liver. The commonly used antiepileptic drugs carbamazepine, phenytoin, valproate, phenobarbital and clonazepam, as well as the convulsant agent t-butylbicyclophosphorothionate (TBPS), picrotoxin and bicuculline did not displace [3H]levetiracetam binding. However, ethosuximide (pKi = 3.5 +/- 0.1), pentobarbital (pKi = 3.8 +/- 0.1), pentylenetetrazole (pKi = 4.1 +/- 0.1) and bemegride (pKi = 5.0 +/- 0.1) competed with [3H]levetiracetam with pKi values comparable to active drug concentrations observed in vivo. Structurally related compounds, including piracetam and aniracetam, also displaced [3H]levetiracetam binding. (S) Stereoisomer homologues of levetiracetam demonstrated a rank order of affinity for [3H]levetiracetam binding in correlation with their

  15. An experimentally based computer search identifies unstructured membrane-binding sites in proteins: application to class I myosins, PAKS, and CARMIL.

    PubMed

    Brzeska, Hanna; Guag, Jake; Remmert, Kirsten; Chacko, Susan; Korn, Edward D

    2010-02-19

    Programs exist for searching protein sequences for potential membrane-penetrating segments (hydrophobic regions) and for lipid-binding sites with highly defined tertiary structures, such as PH, FERM, C2, ENTH, and other domains. However, a rapidly growing number of membrane-associated proteins (including cytoskeletal proteins, kinases, GTP-binding proteins, and their effectors) bind lipids through less structured regions. Here, we describe the development and testing of a simple computer search program that identifies unstructured potential membrane-binding sites. Initially, we found that both basic and hydrophobic amino acids, irrespective of sequence, contribute to the binding to acidic phospholipid vesicles of synthetic peptides that correspond to the putative membrane-binding domains of Acanthamoeba class I myosins. Based on these results, we modified a hydrophobicity scale giving Arg- and Lys-positive, rather than negative, values. Using this basic and hydrophobic scale with a standard search algorithm, we successfully identified previously determined unstructured membrane-binding sites in all 16 proteins tested. Importantly, basic and hydrophobic searches identified previously unknown potential membrane-binding sites in class I myosins, PAKs and CARMIL (capping protein, Arp2/3, myosin I linker; a membrane-associated cytoskeletal scaffold protein), and synthetic peptides and protein domains containing these newly identified sites bound to acidic phospholipids in vitro.

  16. Calmodulin binding proteins of the cholinergic electromotor synapse: synaptosomes, synaptic vesicles, receptor-enriched membranes, and cytoskeleton.

    PubMed

    Walker, J H; Stadler, H; Witzemann, V

    1984-02-01

    Calmodulin binding proteins (CBPs) have been identified using a gel overlay technique for fractions isolated from Torpedo electromotor nerve endings. Different fractions possessed characteristic patterns of CBPs. Synaptosomes showed five major CBPs--Mr 220,000, 160,000, 125,000, 55,000, and 51,000. Polypeptides of Mr 55,000 and 51,000 were found in the cytoplasm and the others are membrane-associated. The Triton X-100-insoluble cytoskeleton of synaptosomes was isolated in the presence or absence of calcium. The major CBPs had Mr of 19,000, 18,000, and 16,000. In the presence of calcium, no other CBPs were seen. In the absence of calcium, an Mr 160,000 polypeptide was present in the Triton cytoskeleton. Synaptic vesicles showed CBPs of Mr 160,000, 25,000, and 20,000. Membrane fragments enriched in acetylcholine receptors contained two major CBPs, Mr 160,000 and 125,000, together with a less prominent protein at Mr 26,000. A protein of Mr similar to that of fodrin was present in synaptosomes and acetylcholine receptor membrane fragments, but only in small amounts relative to the other polypeptides observed. The heavy and light chains of clathrin-coated vesicles from pig brain did not bind calmodulin, although strong labelling of an Mr 47,000 polypeptide was found. Results showed that calelectrin does not bind calmodulin. The possible identity of the calmodulin binding proteins is discussed.

  17. Antimicrobial Peptide Potency is Facilitated by Greater Conformational Flexibility when Binding to Gram-negative Bacterial Inner Membranes

    PubMed Central

    Amos, Sarah-Beth T. A.; Vermeer, Louic S.; Ferguson, Philip M.; Kozlowska, Justyna; Davy, Matthew; Bui, Tam T.; Drake, Alex F.; Lorenz, Christian D.; Mason, A. James

    2016-01-01

    The interaction of antimicrobial peptides (AMPs) with the inner membrane of Gram-negative bacteria is a key determinant of their abilities to exert diverse bactericidal effects. Here we present a molecular level understanding of the initial target membrane interaction for two cationic α-helical AMPs that share structural similarities but have a ten-fold difference in antibacterial potency towards Gram-negative bacteria. The binding and insertion from solution of pleurocidin or magainin 2 to membranes representing the inner membrane of Gram-negative bacteria, comprising a mixture of 128 anionic and 384 zwitterionic lipids, is monitored over 100 ns in all atom molecular dynamics simulations. The effects of the membrane interaction on both the peptide and lipid constituents are considered and compared with new and published experimental data obtained in the steady state. While both magainin 2 and pleurocidin are capable of disrupting bacterial membranes, the greater potency of pleurocidin is linked to its ability to penetrate within the bacterial cell. We show that pleurocidin displays much greater conformational flexibility when compared with magainin 2, resists self-association at the membrane surface and penetrates further into the hydrophobic core of the lipid bilayer. Conformational flexibility is therefore revealed as a key feature required of apparently α-helical cationic AMPs for enhanced antibacterial potency. PMID:27874065

  18. Antimicrobial Peptide Potency is Facilitated by Greater Conformational Flexibility when Binding to Gram-negative Bacterial Inner Membranes

    NASA Astrophysics Data System (ADS)

    Amos, Sarah-Beth T. A.; Vermeer, Louic S.; Ferguson, Philip M.; Kozlowska, Justyna; Davy, Matthew; Bui, Tam T.; Drake, Alex F.; Lorenz, Christian D.; Mason, A. James

    2016-11-01

    The interaction of antimicrobial peptides (AMPs) with the inner membrane of Gram-negative bacteria is a key determinant of their abilities to exert diverse bactericidal effects. Here we present a molecular level understanding of the initial target membrane interaction for two cationic α-helical AMPs that share structural similarities but have a ten-fold difference in antibacterial potency towards Gram-negative bacteria. The binding and insertion from solution of pleurocidin or magainin 2 to membranes representing the inner membrane of Gram-negative bacteria, comprising a mixture of 128 anionic and 384 zwitterionic lipids, is monitored over 100 ns in all atom molecular dynamics simulations. The effects of the membrane interaction on both the peptide and lipid constituents are considered and compared with new and published experimental data obtained in the steady state. While both magainin 2 and pleurocidin are capable of disrupting bacterial membranes, the greater potency of pleurocidin is linked to its ability to penetrate within the bacterial cell. We show that pleurocidin displays much greater conformational flexibility when compared with magainin 2, resists self-association at the membrane surface and penetrates further into the hydrophobic core of the lipid bilayer. Conformational flexibility is therefore revealed as a key feature required of apparently α-helical cationic AMPs for enhanced antibacterial potency.

  19. New approach to measure protein binding based on a parallel artificial membrane assay and human serum albumin.

    PubMed

    Lázaro, Elisabet; Lowe, Philip J; Briand, Xavier; Faller, Bernard

    2008-04-10

    We report here a new, label-free approach to measure serum protein binding constants. The assay is able to measure HSA K d values in the milli-molar to micromolar range. The protein is not immobilized on any surface and the assay self-corrects for nonspecific adsorption. No mass balance is required to get accurate binding constants and it is not necessary to wait for equilibrium to extract the binding constant. The assay runs in a 96-well format using commercially available parts and is, therefore, relatively easy to implement and automate. As the chemical membranes used are not water permeable, there is no volume change due to the osmotic pressure and pretreatment (soaking) is not necessary. The concept can potentially be extended to other proteins and could thus serve as a label-free technique for general binding constant measurements.

  20. Binding of Human Lymphotoxin to Target-Cell Membranes and Its Relation to Cell-Mediated Cytodestruction

    PubMed Central

    Hessinger, David A.; Daynes, Raymond A.; Granger, Gale A.

    1973-01-01

    One of the lymphocyte effector molecules produced in vitro by antigen- and mitogen-stimulated lymphocytes is lymphotoxin, which has been proposed to be the cytodestructive mediator in cell-mediated immune reactions. Our in vitro findings suggest that lymphotoxin binds to sites on the plasma membrane of sensitive target cells, and this binding represents the earliest detectable effect in cell destruction induced by lymphotoxin. The kinetics of binding are rapid, and the affinity of lymphotoxin for binding sites is strong. Furthermore, cytodestruction can be localized, for lymphotoxin-coated cells cannot release lymphotoxin to affect adjacent 51Cr-labeled, noncoated cells in mixed cultures. This evidence suggests how a nonspecific soluble cytotoxin could cause highly specific localized tissue destruction. Images PMID:4544711

  1. Eps15 membrane-binding and -bending activity acts redundantly with Fcho1 during clathrin-mediated endocytosis

    PubMed Central

    Wang, Lei; Johnson, Adam; Hanna, Michael; Audhya, Anjon

    2016-01-01

    Clathrin coat assembly on membranes requires cytosolic adaptors and accessory proteins, which bridge triskeleons with the lipid bilayer and stabilize lattice architecture throughout the process of vesicle formation. In Caenorhabditis elegans, the prototypical AP-2 adaptor complex, which is activated by the accessory factor Fcho1 at the plasma membrane, is dispensable during embryogenesis, enabling us to define alternative mechanisms that facilitate clathrin-mediated endocytosis. Here we uncover a synthetic genetic interaction between C. elegans Fcho1 (FCHO-1) and Eps15 (EHS-1), suggesting that they function in a parallel and potentially redundant manner. Consistent with this idea, we find that the FCHO-1 EFC/F-BAR domain and the EHS-1 EH domains exhibit highly similar membrane-binding and -bending characteristics in vitro. Furthermore, we demonstrate a critical role for EHS-1 when FCHO-1 membrane-binding and -bending activity is specifically eliminated in vivo. Taken together, our data highlight Eps15 as an important membrane-remodeling factor, which acts in a partially redundant manner with Fcho proteins during the earliest stages of clathrin-mediated endocytosis. PMID:27385343

  2. 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.

  3. Global analysis of steady-state energy transfer measurements in membranes: resolution of structural and binding parameters.

    PubMed

    Domanov, Yegor A; Gorbenko, Galina P; Molotkovsky, Julian G

    2004-01-01

    A method has been developed allowing structural and binding parameters to be recovered by global analysis of two-dimensional array of steady-state RET data in the special case where energy acceptors distribute between aqueous and lipid phases while donors are embedded in the membrane at a known depth. To test the validity of this approach, correlation and error analyses have been performed using simulated data. To exemplify the method application to the membrane studies, energy transfer from anthrylvinyl-labeled phosphatidylcholine incorporated into mixed phosphatidylcholine/cardiolipin unilamellar vesicles to heme group of cytochrome c is analyzed.

  4. Androgens and hair growth.

    PubMed

    Randall, Valerie Anne

    2008-01-01

    Hair's importance in human communication means that abnormalities like excess hair in hirsutism or hair loss in alopecia cause psychological distress. Androgens are the main regulator of human hair follicles, changing small vellus follicles producing tiny, virtually invisible hairs into larger intermediate and terminal follicles making bigger, pigmented hairs. The response to androgens varies with the body site as it is specific to the hair follicle itself. Normally around puberty, androgens stimulate axillary and pubic hair in both sexes, plus the beard, etc. in men, while later they may also inhibit scalp hair growth causing androgenetic alopecia. Androgens act within the follicle to alter the mesenchyme-epithelial cell interactions, changing the length of time the hair is growing, the dermal papilla size and dermal papilla cell, keratinocyte and melanocyte activity. Greater understanding of the mechanisms of androgen action in follicles should improve therapies for poorly controlled hair disorders like hirsutism and alopecia.

  5. 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.

  6. Structural basis for androgen specificity and oestrogen synthesis in human aromatase.

    PubMed

    Ghosh, Debashis; Griswold, Jennifer; Erman, Mary; Pangborn, Walter

    2009-01-08

    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(2), 1 mol of NADPH, and coupling with its redox partner cytochrome P450 reductase, aromatase converts androstenedione, testosterone and 16alpha-hydroxytestosterone to oestrone, 17beta-oestradiol and 17beta,16alpha-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.

  7. Modulation of [(35)S]GTPgammaS binding to chinese hamster ovary cell membranes by D(2(short)) dopamine receptors.

    PubMed

    Terasmaa, A; Finnman, U B; Owman, C; Ferré, S; Fuxe, K; Rinken, A

    2000-02-18

    Rat dopamine D(2short) expressed in Chinese hamster ovary (CHO) cells were characterized by means of activation of [(35)S]-guanosine 5'-O-(gamma-thiotriphosphate) ([(35)S]GTPgammaS) binding and inhibition of [(3)H]raclopride binding. Among 18 dopaminergic ligands studied dopamine, NPA, apomorphine and quinpirole were full agonists in activation of [(35)S]GTPgammaS binding, while seven ligands were partial agonists with efficacies from 16 to 69% of the effect of dopamine and seven ligands were antagonists having no effect on the basal level of [(35)S]GTPgammaS binding, but inhibited dopamine-dependent activation in a dose-response manner. Despite the different efficacies, the potencies of all 18 ligands to modulate [(35)S]GTPgammaS binding revealed a good correlation with their potencies to inhibit [(3)H]raclopride binding in the CHO cell membranes. This indicates that the binding of the ligand to the receptor determines its potency, but has no direct correlation with its intrinsic activity.

  8. C601S mutation in the androgen receptor results in partial loss of androgen function.

    PubMed

    Singh, Rajender; Singh, Pooja; Gupta, Nalini J; Chakrabarty, Baidyanath; Singh, Lalji; Thangaraj, Kumarasamy

    2010-11-01

    The present study was undertaken on a case of partial androgen insensitivity syndrome to look at the etiology of the disorder. The patient exhibited a female phenotype despite 46,XY chromosome complement. Direct DNA sequencing of coding region of the androgen receptor gene in this case revealed a 2329G>C substitution (cDNA sequence reference) in exon 3 of the gene. The substitution resulted in replacement of Cys with Ser at codon 601 of the ligand-binding domain of the protein. Analyses on 200 control samples revealed absence of this substitution(s). In vitro assays were done using COS-1 cells. The mutation resulted in partial (∼40%) loss of ligand-binding and significant (∼70%) loss of downstream transactivation function. The mutation was absent in the controls. The findings are particularly interesting since another substitution at the same codon (TGC-TTC) has been reported in association with complete androgen insensitivity syndrome.

  9. Progestin, estrogen and androgen G-protein coupled receptors in fish gonads.

    PubMed

    Thomas, Peter; Dressing, Gwen; Pang, Yefei; Berg, Hakan; Tubbs, Christopher; Benninghoff, Abby; Doughty, Kelly

    2006-04-01

    The identities of the membrane receptors mediating the majority of rapid, cell surface-initiated, nongenomic (i.e. nonclassical) steroid actions described to date are unclear. Two novel 7-transmembrane spanning proteins, representing two distinct classes of steroid membrane receptors, membrane progestin receptor alpha (mPRalpha) and a membrane estrogen receptor (mER), GPR30, have recently been identified in several vertebrate species. Evidence that both receptors activate G-proteins and function as G-protein coupled receptors (GPCRs) is briefly reviewed. New data on progestin actions on fish gametes suggest a widespread involvement of mPRalpha in oocyte maturation and sperm hyperactivity in this vertebrate group. Information on the second messenger pathways activated upon estrogen binding to a membrane estrogen receptor in croaker gonads and preliminary evidence for the presence of a GPR30-like protein in fish gonads are discussed. Finally, initial characterization of the ligand binding, G-protein activation and molecular size of a membrane androgen receptor (mAR) in croaker ovaries suggests the presence of a third unique steroid receptor in fish gonads that also may function as a GPCR.

  10. 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

  11. Dynamic light scattering analysis of SNARE-driven membrane fusion and the effects of SNARE-binding flavonoids.

    PubMed

    Yang, Yoosoo; Heo, Paul; Kong, Byoungjae; Park, Jun-Bum; Jung, Young-Hun; Shin, Jonghyeok; Jeong, Cherlhyun; Kweon, Dae-Hyuk

    2015-10-02

    Soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins generate energy required for membrane fusion. They form a parallelly aligned four-helix bundle called the SNARE complex, whose formation is initiated from the N terminus and proceeds toward the membrane-proximal C terminus. Previously, we have shown that this zippering-like process can be controlled by several flavonoids that bind to the intermediate structures formed during the SNARE zippering. Here, our aim was to test whether the fluorescence resonance energy transfer signals that are observed during the inner leaflet mixing assay indeed represent the hemifused vesicles. We show that changes in vesicle size accompanying the merging of bilayers is a good measure of progression of the membrane fusion. Two merging vesicles with the same size D in diameter exhibited their hydrodynamic diameters 2D + d (d, intermembrane distance), 2D and 2D as membrane fusion progressed from vesicle docking to hemifusion and full fusion, respectively. A dynamic light scattering assay of membrane fusion suggested that myricetin stopped membrane fusion at the hemifusion state, whereas delphinidin and cyanidin prevented the docking of the vesicles. These results are consistent with our previous findings in fluorescence resonance energy transfer assays.

  12. 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

  13. 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.

  14. 2-(/sup 125/I)iodomelatonin binding sites in hamster brain membranes: pharmacological characteristics and regional distribution

    SciTech Connect

    Duncan, M.J.; Takahashi, J.S.; Dubocovich, M.L.

    1988-05-01

    Studies in a variety of seasonally breeding mammals have shown that melatonin mediates photoperiodic effects on reproduction. Relatively little is known, however, about the site(s) or mechanisms of action of this hormone for inducing reproductive effects. Although binding sites for (3H)melatonin have been reported previously in bovine, rat, and hamster brain, the pharmacological selectivity of these sites was never demonstrated. In the present study, we have characterized binding sites for a new radioligand, 2-(125I)iodomelatonin, in brains from a photoperiodic species, the Syrian hamster. 2-(125I)Iodomelatonin labels a high affinity binding site in hamster brain membranes. Specific binding of 2-(125I)iodomelatonin is rapid, stable, saturable, and reversible. Saturation studies demonstrated that 2-(125I)iodomelatonin binds to a single class of sites with an affinity constant (Kd) of 3.3 +/- 0.5 nM and a total binding capacity (Bmax) of 110.2 +/- 13.4 fmol/mg protein (n = 4). The Kd value determined from kinetic analysis (3.1 +/- 0.9 nM; n = 5) was very similar to that obtained from saturation experiments. Competition experiments showed that the relative order of potency of a variety of indoles for inhibition of 2-(125I)iodomelatonin binding site to hamster brain membranes was as follows: 6-chloromelatonin greater than or equal to 2-iodomelatonin greater than N-acetylserotonin greater than or equal to 6-methoxymelatonin greater than or equal to melatonin greater than 6-hydroxymelatonin greater than or equal to 6,7-dichloro-2-methylmelatonin greater than 5-methoxytryptophol greater than 5-methoxytryptamine greater than or equal to 5-methoxy-N,N-dimethyltryptamine greater than N-acetyltryptamine greater than serotonin greater than 5-methoxyindole (inactive).

  15. L712V mutation in the androgen receptor gene causes complete androgen insensitivity syndrome due to severe loss of androgen function.

    PubMed

    Rajender, Singh; Gupta, Nalini J; Chakrabarty, Baidyanath; Singh, Lalji; Thangaraj, Kumarasamy

    2013-12-11

    Inability to respond to the circulating androgens is named as androgen insensitivity syndrome (AIS). Mutations in the androgen receptor (AR) gene are the most common cause of AIS. A cause and effect relationship between some of these mutations and the AIS phenotype has been proven by in vitro studies. Several other mutations have been identified, but need to be functionally validated for pathogenicity. Screening of the AR mutations upon presumptive diagnosis of AIS is recommended. We analyzed a case of complete androgen insensitivity syndrome (CAIS) for mutations in the AR gene. Sequencing of the entire coding region revealed C>G mutation (CTT-GTT) at codon 712 (position according to the NCBI database) in exon 4 of the gene, resulting in replacement of leucine with valine in the ligand-binding domain of the AR protein. No incidence of this mutation was observed in 230 normal male individuals analyzed for comparison. In vitro androgen binding and transactivation assays using mutant clone showed approximately 71% loss of ligand binding and about 76% loss of transactivation function. We conclude that CAIS in this individual was due to L712V substitution in the androgen receptor protein.

  16. Characterization of a multiple endogenously expressed adenosine triphosphate-binding cassette transporters using nuclear and cellular membrane affinity chromatography columns.

    PubMed

    Habicht, K-L; Singh, N S; Khadeer, M A; Shimmo, R; Wainer, I W; Moaddel, R

    2014-04-25

    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 LN-229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN-229)) and (CMAC(LN-229)), respectively. Pgp, MRP1 and BCRP transporters co-immobilized on both columns were 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 eight 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(LN-229) column and decreased it (-5%) on the NMAC(LN-229), 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.

  17. Resolving the challenge of measuring ligand binding to membrane proteins by combining analytical ultracentrifugation and light scattering photometry.

    PubMed

    Doran, J D; Mohanty, A K; Fox, T

    2012-01-01

    Membrane proteins are attractive therapeutic targets, however the presence of detergents complicates biophysical binding measurements. Difficulties in determining quantitative dissociation constants for problematic membrane proteins were addressed by combining analytical ultracentrifugation and classical light scattering techniques. Validation of the algorithm used to calculate dissociation constants from sedimentation equilibrium experiments was demonstrated by analyzing binding data of the inhibitor Y-27632 to rho-kinase (ROCK). Kd's of 1.3 ± 0.7 and 52 ± 27 µM were calculated for ROCK constructs (S6-R415) and (M71-E379) respectively, consistent with previously published Ki's of 1.4 ± 0.1 and > 30 µM. Extension of the algorithm to membrane proteins required the collection of light scattering data to determine the partial specific volume, ν, for the membrane protein-detergent complex. Vitamin B12 binding to the bacterial protein btuB in octyl β-D-glucopyranoside (β-OG) illustrates the applicability of the method. A ν of 0.781 ml/g was determined for the btuB-β-OG complex. Incorporating this value into the algorithm generated a Kd of 7.0 ± 1.5 µM for the vitamin B12-btuB affinity. A Kd of 9.7 ± 2.7 µM was determined by equilibrium dialysis under similar experimental conditions. Successfully applying AUC to quantifying small-molecule ligand affinities to membrane proteins represents a significant advance to the field.

  18. 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

  19. 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

  20. 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.

  1. Sugar binding induces the same global conformational change in purified LacY as in the native bacterial membrane.

    PubMed

    Nie, Yiling; Kaback, H Ronald

    2010-05-25

    Many independent lines of evidence indicate that the lactose permease of Escherichia coli (LacY) is highly dynamic and that sugar binding causes closing of a large inward-facing cavity with opening of a wide outward-facing hydrophilic cavity. Therefore, lactose/H(+) symport catalyzed by LacY very likely involves a global conformational change that allows alternating access of single sugar- and H(+)-binding sites to either side of the membrane (the alternating access model). The x-ray crystal structures of LacY, as well as the majority of spectroscopic studies, use purified protein in detergent micelles. By using site-directed alkylation, we now demonstrate that sugar binding induces virtually the same global conformational change in LacY whether the protein is in the native bacterial membrane or is solubilized and purified in detergent. The results also indicate that the x-ray crystal structure reflects the structure of wild-type LacY in the native membrane in the absence of sugar.

  2. Autoimmune anti-androgen-receptor antibodies in human serum.

    PubMed Central

    Liao, S; Witte, D

    1985-01-01

    Circulating autoantibodies to human and rat androgen receptors are present at high titers in the blood sera of some patients with prostate diseases. The antibodies from some serum samples were associated with a purified IgG fraction and interacted with the 3.8S cytosolic androgen-receptor complexes of rat ventral prostate to form 9- to 12S units. Other serum samples, however, formed 14- to 19S units, suggesting that other immunoglobulins might be involved. In the presence of an anti-human immunoglobulin as a second antibody, the androgen-receptor-antibody complexes could be immunoprecipitated. The antibodies interacted with the nuclear and the cytosolic androgen-receptor complexes, either the DNA-binding or the nonbinding form, but not with receptors for estradiol, progestin, or dexamethasone from a variety of sources. Human testosterone/estradiol-binding globulin, rat epididymal androgen-binding protein, or rat prostate alpha-protein (a nonreceptor steroid-binding protein) also did not interact with the antibodies to form immunoprecipitates. About 37% of male and 3% of female serum samples screened had significant antibody titer. The chance of finding serum with a high titer is much better in males older than 66 years than in the younger males or females at all ages. The presence of the high-titer antibodies may make it possible to prepare monoclonal antibodies to androgen receptors without purification of the receptors for immunization. PMID:3866227

  3. 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

    2016-09-13

    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.

  4. Tooth Enamel Protein Amelogenin Binds to Ameloblast Cell Membrane-Mimicking Vesicles via its N-terminus

    PubMed Central

    LOKAPPA, SOWMYA BEKSHE; CHANDRABABU, KARTHIK BALAKRISHNA; MORADIAN-OLDAK, JANET

    2015-01-01

    We have recently reported that the extracellular enamel protein amelogenin has affinity to interact with phospholipids and proposed that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities. Here, in order to identify the liposome-interacting domains of amelogenin we designed four different amelogenin mutants containing only a single tryptophan at positions 25, 45, 112 and 161. Circular dichroism studies of the mutants confirmed that they are structurally similar to the wild-type amelogenin. Utilizing the intrinsic fluorescence of single tryptophan residues and fluorescence resonance energy transfer FRET, we analyzed the accessibility and strength of their binding with an ameloblast cell membrane-mimicking model membrane (ACML) and a negatively charged liposome used as a membrane model. We found that amelogenin has membrane-binding ability mainly via its N-terminal, close to residues W25 and W45. Significant blue shift was also observed in the fluorescence of a N-terminal peptide following addition of liposomes. We suggest that, among other mechanisms, enamel malformation in cases of Amelogenesis Imperfecta (AI) with mutations at the N-terminal may be the result of defective amelogenin-cell interactions. PMID:26188506

  5. Plasma membrane fatty acid-binding protein and mitochondrial glutamic-oxaloacetic transaminase of rat liver are related

    SciTech Connect

    Berk, P.D.; Potter, B.J.; Sorrentino, D.; Zhou, S.L.; Isola, L.M.; Stump, D.; Kiang, C.L.; Thung, S. ); Wada, H.; Horio, Y. )

    1990-05-01

    The hepatic plasma membrane fatty acid-binding protein (h-FABP{sub PM}) and the mitochondrial isoenzyme of glutamic-oxaloacetic transaminase (mGOT) of rat liver have similar amino acid compositions and identical amino acid sequences for residues 3-24. Both proteins migrate with an apparent molecular mass of 43 kDa on SDS/polyacrylamide gel electrophoresis, have a similar pattern of basic charge isomers on isoelectric focusing, are eluted similarly from four different high-performance liquid chromatographic columns, have absorption maxima at 435 nm under acid conditions and 354 nm at pH 8.3, and bind oleate. Sinusoidally enriched liver plasma membranes and purified h-FABP{sub PM} have GOT enzymatic activity. Monospecific rabbit antiserum against h-FABP{sub PM} reacts on Western blotting with mGOT, and vice versa. Antisera against both proteins produce plasma membrane immunofluorescence in rat hepatocytes and selectively inhibit the hepatocellular uptake of ({sup 3}H)oleate but not that of ({sup 35}S)sulfobromophthalein or ({sup 14}C)taurocholate. The inhibition of oleate uptake produced by anti-h-FABP{sub PM} can be eliminated by preincubation of the antiserum with mGOT; similarly, the plasma membrane immunofluorescence produced by either antiserum can be eliminated by preincubation with the other antigen. These data suggest that h-FABP{sub PM} and mGOT are closely related.

  6. 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

  7. Binding affinity of amyloid oligomers to cellular membranes is a generic indicator of cellular dysfunction in protein misfolding diseases

    PubMed Central

    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 Ca2+ 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

  8. Analysis of (/sup 3/H) Kainic acid binding with rat and Frog brain membranes

    SciTech Connect

    Zharkovskii, A.M.; Zharkovskaya, T.A.

    1985-10-01

    This paper analyzes the binding of (H 3)-KA with membrances in vitro and the effect of various neuroactive amino acids, suggested as endogenous ligands for binding sites of (H 3)-KA, on binding. Experiments were carried out on male albino rats and on winter frogs. Choice of the frog's brain was determined by the high density of high-affinity binding sites of (H 3)-KA. The concentrations of substances inhibiting binding (H 3)-KA by 50% were calculated by logit-probit analysis, and inhibition constants were also calculated. It is shown that although L-glutamate and folic acid inhibit binding of (H 3)-KA, they do not satisfy the criteria to be met by endogenous ligands, and this inhibition of binding is noncompetitive in character. This suggests that KA binding sites and glutamate receptors are not identical, although they may perhaps be subunits of a single complex.

  9. Differential Mechanisms of Androgen Resistance

    DTIC Science & Technology

    2007-12-01

    Tincello, DG, Shalet, SM and Wu FC. Point mutatons detected in the androgen receptor gene of three men with partial androgen insensitivity syndrome . Clin...with androgen insensitivity syndrome (Turek-Plewa et al, 2006, Kohler, et al, 2005, Komori et al, 1997, Brown et al 1992, Saunders et al 1992... Androgen insensitivity syndrome is often associated with the decreased androgen receptor activity. The identification mutations in our xenografted

  10. ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification

    PubMed Central

    Gulati, Sonia; Balderes, Dina; Kim, Christine; Guo, Zhongmin A.; Wilcox, Lisa; Area-Gomez, Estela; Snider, Jamie; Wolinski, Heimo; Stagljar, Igor; Granato, Juliana T.; Ruggles, Kelly V.; DeGiorgis, Joseph A.; Kohlwein, Sepp D.; Schon, Eric A.; Sturley, Stephen L.

    2015-01-01

    A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53–36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.—Gulati, S., Balderes, D., Kim, C., Guo, Z. A., Wilcox, L., Area-Gomez, E., Snider, J., Wolinski, H., Stagljar, I., Granato, J. T., Ruggles, K. V., DeGiorgis, J. A., Kohlwein, S. D., Schon, E. A., Sturley, S. L. ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification. PMID:26220175

  11. 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

  12. Variation of the Detergent-Binding Capacity and Phospholipid Content of Membrane Proteins When Purified in Different Detergents

    PubMed Central

    Ilgü, Hüseyin; Jeckelmann, Jean-Marc; Gachet, María Salomé; Boggavarapu, Rajendra; Ucurum, Zöhre; Gertsch, Jürg; Fotiadis, Dimitrios

    2014-01-01

    Purified membrane proteins are ternary complexes consisting of protein, lipid, and detergent. Information about the amounts of detergent and endogenous phospholipid molecules bound to purified membrane proteins is largely lacking. In this systematic study, three model membrane proteins of different oligomeric states were purified in nine different detergents at commonly used concentrations and characterized biochemically and biophysically. Detergent-binding capacities and phospholipid contents of the model proteins were determined and compared. The insights on ternary complexes obtained from the experimental results, when put into a general context, are summarized as follows. 1), The amount of detergent and 2) the amount of endogenous phospholipids bound to purified membrane proteins are dependent on the size of the hydrophobic lipid-accessible protein surface areas and the physicochemical properties of the detergents used. 3), The size of the detergent and lipid belt surrounding the hydrophobic lipid-accessible surface of purified membrane proteins can be tuned by the appropriate choice of detergent. 4), The detergents n-nonyl-β-D-glucopyranoside and Cymal-5 have exceptional delipidating effects on ternary complexes. 5), The types of endogenous phospholipids bound to membrane proteins can vary depending on the detergent used for solubilization and purification. 6), Furthermore, we demonstrate that size-exclusion chromatography can be a suitable method for estimating the molecular mass of ternary complexes. The findings presented suggest a strategy to control and tune the numbers of detergent and endogenous phospholipid molecules bound to membrane proteins. These two parameters are potentially important for the successul crystallization of membrane proteins for structure determination by crystallographic approaches. PMID:24739165

  13. Variation of the detergent-binding capacity and phospholipid content of membrane proteins when purified in different detergents.

    PubMed

    Ilgü, Hüseyin; Jeckelmann, Jean-Marc; Gachet, María Salomé; Boggavarapu, Rajendra; Ucurum, Zöhre; Gertsch, Jürg; Fotiadis, Dimitrios

    2014-04-15

    Purified membrane proteins are ternary complexes consisting of protein, lipid, and detergent. Information about the amounts of detergent and endogenous phospholipid molecules bound to purified membrane proteins is largely lacking. In this systematic study, three model membrane proteins of different oligomeric states were purified in nine different detergents at commonly used concentrations and characterized biochemically and biophysically. Detergent-binding capacities and phospholipid contents of the model proteins were determined and compared. The insights on ternary complexes obtained from the experimental results, when put into a general context, are summarized as follows. 1), The amount of detergent and 2) the amount of endogenous phospholipids bound to purified membrane proteins are dependent on the size of the hydrophobic lipid-accessible protein surface areas and the physicochemical properties of the detergents used. 3), The size of the detergent and lipid belt surrounding the hydrophobic lipid-accessible surface of purified membrane proteins can be tuned by the appropriate choice of detergent. 4), The detergents n-nonyl-β-D-glucopyranoside and Cymal-5 have exceptional delipidating effects on ternary complexes. 5), The types of endogenous phospholipids bound to membrane proteins can vary depending on the detergent used for solubilization and purification. 6), Furthermore, we demonstrate that size-exclusion chromatography can be a suitable method for estimating the molecular mass of ternary complexes. The findings presented suggest a strategy to control and tune the numbers of detergent and endogenous phospholipid molecules bound to membrane proteins. These two parameters are potentially important for the successul crystallization of membrane proteins for structure determination by crystallographic approaches.

  14. 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

  15. A dual antibacterial mechanism involved in membrane disruption and DNA binding of 2R,3R-dihydromyricetin from pine needles of Cedrus deodara against Staphylococcus aureus.

    PubMed

    Wu, Yanping; Bai, Jinrong; Zhong, Kai; Huang, Yina; Gao, Hong

    2017-03-01

    The antibacterial activity and mechanism of 2R,3R-dihydromyricetin (DMY) against Staphylococcus aureus were investigated. The minimum inhibitory concentration of DMY against S. aureus was 0.125mg/ml, and the growth inhibitory assay also revealed that DMY showed a potent antibacterial activity against S. aureus. Massive nucleotide leakage and flow cytometric analysis demonstrated that DMY disrupted the membrane integrity of S. aureus. Morphological changes and membrane hyperpolarization of S. aureus cells treated with DMY further suggested that DMY destroyed cell membrane. Meanwhile, DMY probably interacted with membrane lipids and proteins, causing a significant reduction in membrane fluidity and changes in conformation of membrane protein. Moreover, DMY could interact with S. aureus DNA through the groove binding mode. Overall, the results suggested that DMY could be applied as a candidate for the development of new food preservatives as it achieved bactericidal activity by damaging cell membrane and binding to intracellular DNA.

  16. Do androgens influence hair growth by altering the paracrine factors secreted by dermal papilla cells?

    PubMed

    Randall, V A; Hibberts, N A; Thornton, M J; Merrick, A E; Hamada, K; Kato, S; Jenner, T J; de Oliveira, I; Messenger, A G

    2001-01-01

    Androgens regulate many aspects of human hair growth in both sexes. After puberty they transform tiny vellus follicles in many areas, e.g. the face, to terminal ones producing long, thick, pigmented hairs. In genetically predisposed individuals, androgens also cause the reverse transformation of terminal scalp follicles into vellus ones, causing balding. In the current hypothesis for androgen action, androgens control most follicular cells indirectly acting via the mesenchyme-derived dermal papilla which regulates many aspects of follicular activity. In this model androgens binding to androgen receptors in dermal papilla cells alter their production of regulatory molecules which influence other follicular components; these molecules may be soluble paracrine factors and/or extracellular matrix proteins. This hypothesis is supported by immunohistochemical localisation of androgen receptors in dermal papilla cell nuclei and the demonstrations that androgen receptor content and testosterone metabolism patterns of cultured dermal papilla cells from various body sites reflect hair growth in androgen-insensitivity syndromes. The next question is whether androgens alter the paracrine factors secreted by dermal papilla cells. Cultured dermal papilla cells do release soluble, proteinaceous factors into their media which stimulate the growth of keratinocytes and other dermal papilla cells. This mitogenic potential can cross species from humans to rodents. Importantly, testosterone in vitro stimulates the mitogenic potential of beard cells, but in contrast inhibits production by balding scalp cells reflecting their in vivo androgenic responses. Since androgens in vitro do alter the secretion of paracrine factors the current focus lies in identifying specific factors produced, e.g. IGF-I and stem cell factor (SCF), using ELISA and RT-PCR, and comparing their expression in cells from follicles with varying responses to androgens in vivo or under androgen stimulation in vitro

  17. Uptake and binding of /sup 125/I-calmodulin by isolated rat renal brush border membrane vesicles

    SciTech Connect

    Meezan, E.; Elgavish, A.; Wallace, R.W.

    1986-05-01

    The authors have investigated the interaction of /sup 125/I-calmodulin with isolated rat renal brush border membrane vesicles (BBV) using an experimental protocol which allows us to distinguish between ligand binding to the outside of the vesicles vs. uptake and possible binding to the vesicle interior. By examining the association of /sup 125/I-calmodulin with BBV as a function of medium osmolarity (300-1100 mosm) to alter intravesicular space, virtually all ligand interaction with BBV was found to represent uptake of intact /sup 125/I-calmodulin into the intravesicular space. Uptake appeared specific by the following criteria: (1) it was largely calcium dependent (2) it was inhibited in a dose dependent fashion by calmodulin and the homologous protein troponin C, but not by unrelated proteins (lysozyme, cytochrome C, insulin) (3) it was inhibited by known calmodulin antagonists (calmidazolium, mellitin, trifluoperazine). Calmodulin uptake may be followed by binding of /sup 125/I-calmodulin to intravesicular BBV proteins; calmodulin-binding proteins in BBV with molecular weights of 143K, 118K, 50K, 47.5K, 46.5K and 35K were identified by Western blotting techniques. The specific association of /sup 125/I-calmodulin with isolated BBV is of interest in regard to the possible role of this calcium regulatory protein in the protein reabsorptive and ion transport functions of this renal tubular membrane fraction.

  18. Bacillus thuringiensis Cry34Ab1/Cry35Ab1 Interactions with Western Corn Rootworm Midgut Membrane Binding Sites

    PubMed Central

    Li, Huarong; Olson, Monica; Lin, Gaofeng; Hey, Timothy; Tan, Sek Yee; Narva, Kenneth E.

    2013-01-01

    Background Bacillus thuringiensis (Bt) Cry34Ab1/Cry35Ab1 are binary insecticidal proteins that are co-expressed in transgenic corn hybrids for control of western corn rootworm, Diabrotica virgifera virgifera LeConte. Bt crystal (Cry) proteins with limited potential for field-relevant cross-resistance are used in combination, along with non-transgenic corn refuges, as a strategy to delay development of resistant rootworm populations. Differences in insect midgut membrane binding site interactions are one line of evidence that Bt protein mechanisms of action differ and that the probability of receptor-mediated cross-resistance is low. Methodology/Principal Findings Binding site interactions were investigated between Cry34Ab1/Cry35Ab1 and coleopteran active insecticidal proteins Cry3Aa, Cry6Aa, and Cry8Ba on western corn rootworm midgut brush border membrane vesicles (BBMV). Competitive binding of radio-labeled proteins to western corn rootworm BBMV was used as a measure of shared binding sites. Our work shows that 125I-Cry35Ab1 binds to rootworm BBMV, Cry34Ab1 enhances 125I-Cry35Ab1 specific binding, and that 125I-Cry35Ab1 with or without unlabeled Cry34Ab1 does not share binding sites with Cry3Aa, Cry6Aa, or Cry8Ba. Two primary lines of evidence presented here support the lack of shared binding sites between Cry34Ab1/Cry35Ab1 and the aforementioned proteins: 1) No competitive binding to rootworm BBMV was observed for competitor proteins when used in excess with 125I-Cry35Ab1 alone or combined with unlabeled Cry34Ab1, and 2) No competitive binding to rootworm BBMV was observed for unlabeled Cry34Ab1 and Cry35Ab1, or a combination of the two, when used in excess with 125I-Cry3Aa, or 125I-Cry8Ba. Conclusions/Significance Combining two or more insecticidal proteins active against the same target pest is one tactic to delay the onset of resistance to either protein. We conclude that Cry34Ab1/Cry35Ab1 are compatible with Cry3Aa, Cry6Aa, or Cry8Ba for deployment as insect

  19. 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

  20. 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.

  1. 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.

  2. The binding of cyanide to cytochrome d in intact cells, spheroplasts, membrane fragments and solubilized enzyme from Salmonella typhimurium.

    PubMed

    Keyhani, E; Minai-Tehrani, D

    2001-07-02

    This investigation focused on the kinetics of cyanide binding to oxidized and reduced cytochrome d in Salmonella typhimurium intact cells, spheroplasts, membrane fragments and solubilized enzyme, and on the effect of pH on this binding. Cyanide bound to the oxidized form of cytochrome d under all experimental conditions, inducing a trough at 649 nm in the oxidized-cyanide-minus-oxidized difference absorption spectra. V(max) of cyanide binding to oxidized cytochrome d at pH 7.0 was 14.0+/-2.0 pmol/min/mg protein (prot.) in intact cells, 37.0+/-3.5 pmol/min/mg prot. in spheroplasts, 125.0+/-6.0 pmol/min/mg prot. in membrane fragments, and 538.0+/-8.5 pmol/min/mg prot. in solubilized cytochrome d. The pseudo-first order rate constants were 0.004 s(-1) for intact cells, 0.005 s(-1) for spheroplasts, 0.007 s(-1) for membrane fragments and 0.025 s(-1) for the solubilized enzyme. The V(max) value was highest at pH 7.0 for intact cells and solubilized cytochrome d and at pH 8.0 for both spheroplasts and membrane fragments. The K(s) of binding at pH 7.0 was around 4 mM in intact cells, spheroplasts and membrane fragments, but was 10.5 mM in solubilized cytochrome d. This difference between the K(s) values suggested a change in conformation, upon solubilization, leading to a decrease in the affinity of cyanide for the solubilized enzyme. The K(s) value was nearly the same at all pH investigated (pH 5-10). Cyanide was found to also bind to the reduced form of cytochrome d in membrane fragments (K(s)=18+/-3 mM, V(max)=377+/-28 pmol/min/mg prot. at pH 7) and the solubilized enzyme (K(s)=18+/-1.2 mM, V(max)=649+/-45 pmol/min/mg prot. at pH 7) with a lower affinity of cyanide for the reduced cytochrome d than for the oxidized enzyme. Pseudo-first order rate constants were 0.025 s(-1) and 0.042 s(-1) respectively for membrane fragments and solubilized enzyme. The value of V(max) for cyanide binding to the reduced cytochrome d, whether membrane-bound or solubilized, increased

  3. ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification.

    PubMed

    Gulati, Sonia; Balderes, Dina; Kim, Christine; Guo, Zhongmin A; Wilcox, Lisa; Area-Gomez, Estela; Snider, Jamie; Wolinski, Heimo; Stagljar, Igor; Granato, Juliana T; Ruggles, Kelly V; DeGiorgis, Joseph A; Kohlwein, Sepp D; Schon, Eric A; Sturley, Stephen L

    2015-11-01

    A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53-36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.

  4. ``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.

  5. Proteomic Characterization of Pig Sperm Anterior Head Plasma Membrane Reveals Roles of Acrosomal Proteins in ZP3 Binding.

    PubMed

    Kongmanas, Kessiri; Kruevaisayawan, Hathairat; Saewu, Arpornrad; Sugeng, Clarissa; Fernandes, Jason; Souda, Puneet; Angel, Jonathan B; Faull, Kym F; Aitken, R John; Whitelegge, Julian; Hardy, Daniel; Berger, Trish; Baker, Mark A; Tanphaichitr, Nongnuj

    2015-02-01

    The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm-ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non-capacitated and capacitated sperm. The results revealed that ZP-binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far-western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non-capacitated and capacitated sperm, but the complexes (∼750-1300 kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome-intact sperm during capacitation. Co-immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with anti-zonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm-ZP binding during capacitation.

  6. Characterization of [11C]vorozole binding in ovarian tissue in rats throughout estrous cycle in association with conversion of androgens to estrogens in vivo and in vitro.

    PubMed

    Kirilovas, Dmitrijus; Naessen, Tord; Bergström, Mats; Bergström-Petterman, Elizabeth; Carlström, Kjell; Långström, Bengt

    2003-12-01

    Estrogen levels vary in a cyclic fashion during the rat estrous cycle, reaching peak concentrations during proestrus. Previously, it was suggested that the preovulatory peak in estrogen production in rats in vivo is regulated by other control mechanisms than concentration of precursor and amount of aromatase enzyme, changing the specific activity of the enzyme. To explore this hypothesis, ovarian binding of [11C]vorozole in vivo and in vitro, representing the amount of active aromatase, and conversion activity of ovarian homogenate were assayed together with serum androstenedione (A4) and estradiol-17beta (E2) levels during the estrous cycle in rats. The reducing ovarian [11C]vorozole binding in vivo from proestrus +4 up to +8h might indicate that the ovarian aromatase is blocked, probably to prevent premature increase of E2 levels. Thereafter (between proestrus +9 and +13h), the binding dramatically increases (aromatase enzyme is unblocked), to enable increased E2 synthesis. In addition, during the latter period, serum E2 levels were strongly correlated with serum A4 levels after adjustment for amount of ovarian aromatase (P=0.03), but not with amount of aromatase adjusted for levels of A4 (P=0.13), which might indicate changes in specific activity of the aromatase enzyme. Significant correlation between Kd and serum E2 levels during the same period indicated that aromatase-precursor affinity might be involved in the regulation of the enzyme-specific activity. This conclusion is done assuming that [11C]vorozole binding mimics that of the substrate (A4). The [11C]vorozole in vivo technique keeps auto- and paracrine mechanisms intact, and might therefore yield additional information about biological processes compared with traditional in vitro techniques.

  7. 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

  8. 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

  9. Germline variant FGFR4  p.G388R exposes a membrane-proximal STAT3 binding site.

    PubMed

    Ulaganathan, Vijay K; Sperl, Bianca; Rapp, Ulf R; Ullrich, Axel

    2015-12-24

    Variant rs351855-G/A is a commonly occurring single-nucleotide polymorphism of coding regions in exon 9 of the fibroblast growth factor receptor FGFR4 (CD334) gene (c.1162G>A). It results in an amino-acid change at codon 388 from glycine to arginine (p.Gly388Arg) in the transmembrane domain of the receptor. Despite compelling genetic evidence for the association of this common variant with cancers of the bone, breast, colon, prostate, skin, lung, head and neck, as well as soft-tissue sarcomas and non-Hodgkin lymphoma, the underlying biological mechanism has remained elusive. Here we show that substitution of the conserved glycine 388 residue to a charged arginine residue alters the transmembrane spanning segment and exposes a membrane-proximal cytoplasmic signal transducer and activator of transcription 3 (STAT3) binding site Y(390)-(P)XXQ(393). We demonstrate that such membrane-proximal STAT3 binding motifs in the germline of type I membrane receptors enhance STAT3 tyrosine phosphorylation by recruiting STAT3 proteins to the inner cell membrane. Remarkably, such germline variants frequently co-localize with somatic mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Using Fgfr4 single nucleotide polymorphism knock-in mice and transgenic mouse models for breast and lung cancers, we validate the enhanced STAT3 signalling induced by the FGFR4 Arg388-variant in vivo. Thus, our findings elucidate the molecular mechanism behind the genetic association of rs351855 with accelerated cancer progression and suggest that germline variants of cell-surface molecules that recruit STAT3 to the inner cell membrane are a significant risk for cancer prognosis and disease progression.

  10. Interaction of blood sex steroid-binding globulin-steroid complexes with the plasma membranes of cells of the human decidual endometrium

    SciTech Connect

    Zhuk, N.I.; Avvakumov, G.V.; Strel'chenok, O.A.

    1986-01-10

    The plasma membranes of cells of the decidual tissue specifically bind complexes of the sex steroid-binding globulin (SBG) of the blood with estrogens (estradiol, estriol, estrone) and the pharmacological agent danazol but do not interact with SBG-testosterone and SBG-dihydrotestosterone complexes. The selectivity of the interaction of SBG-steroid complexes with the cell membranes of the decidual tissue confirms the hypothesis of an active role of SBG in the action of steroids on this target tissue.

  11. Androgen insensitivity syndrome.

    PubMed

    Mongan, Nigel P; Tadokoro-Cuccaro, Rieko; Bunch, Trevor; Hughes, Ieuan A

    2015-08-01

    Androgen insensitivity syndrome (AIS) results from androgen receptor dysfunction and is a common cause of disorder of sex development. The AIS phenotype largely depends on the degree of residual androgen receptor (AR) activity. This review describes the molecular action of androgens and the range of androgen receptor gene mutations, essential knowledge to understand the pathogenesis of the complete and partial forms of this syndrome. A multidisciplinary approach is recommended for clinical management from infancy through to adulthood. Hormone replacement therapy is needed following gonadectomy. Patients who choose to retain the gonads are at risk of developing germ cell tumors for which sensitive circulating tumor markers may soon become available. Whilst the contribution of AR dysfunction to complete AIS is well understood, the involvement of the AR and associated proteins as contributors to partial AIS is an area of active research. Disorders of sex development such as AIS which are related to AR dysfunction offer a breadth of manifestations for the clinician to manage and opportunities for further research on the mechanism of androgen action.

  12. Characterization of the Ptychodiscus brevis Polyether Neurotoxin Binding Component in Excitable Membranes

    DTIC Science & Technology

    1986-07-31

    Centruroides suffusus suffusus toxin II to neurotoxin receptoi Site 4 by the brevetoxins. Catterall and Gainer (16) suggested that the brevetoxins bind at a...binding of 1251 Centruroides suffusus suffusus toxin 11 on neurotoxin receptor site 4. Purification of both alpha- and beta-scorpion toxins is...guinguestraitus venom (site 3), or Centruroides sculpturatus venom (site 4) (Fig 13 ). Tritiated brevetoxin binding is slightly enhanced (5-10%) in the presence

  13. Kinetics of Bacillus thuringiensis toxin binding with brush border membrane vesicles from susceptible and resistant larvae of Plutella xylostella.

    PubMed

    Masson, L; Mazza, A; Brousseau, R; Tabashnik, B

    1995-05-19

    An optical biosensor technology based on surface plasmon resonance was used to determine the kinetic rate constants for interactions between the CryIA(c) toxin from Bacillus thuringiensis and brush border membrane vesicles purified from susceptible and resistant larvae of diamondback moth (Plutella xylostella). CryIA(c) association and dissociation rate constants for vesicles from susceptible larvae were determined to be 4.5 x 10(3) M-1 s-1 and 3.2 x 10(-5) s-1, respectively, resulting in a calculated affinity constant of 7 nM. CryIE toxin did not kill susceptible or resistant larvae and did not bind to brush border vesicles. Contrary to expectations based on previous studies of binding in resistant P. xylostella, the binding kinetics for CryIA(c) did not differ significantly between susceptible larvae and those that were resistant to CryIA(c). Determination of the number of CryIA(c) receptors revealed an approximately 3-fold decrease in total CryIA(c) receptor numbers for resistant vesicles. These results suggest that factors other than binding may be altered in our resistant diamondback moth strain. They also support the view that binding is not sufficient for toxicity.

  14. The novel alpha 2-adrenoceptor agonist [3H]mivazerol binds to non-adrenergic binding sites in human striatum membranes that are distinct from imidazoline receptors.

    PubMed

    Flamez, A; Gillard, M; De Backer, J P; Vauquelin, G; Noyer, M

    1997-07-01

    The alpha 2 adrenergic agonist [3H]mivazerol labelled two populations of binding sites in membranes from the human striatum. Forty per cent of the sites labelled by 3 nM [3H]mivazerol corresponded to alpha 2 adrenergic receptors as they displayed a high affinity for (-)-adrenaline and for rauwolscine. The remaining binding was displaced by mivazerol with a pIC50 of 6.5 +/- 0.1. These sites displayed higher affinity for dexmedetomidine (pIC50 = 7.1 +/- 0.1), but much lower affinity for clonidine (pIC50 < 5.0) and for idazoxan (pIC50 = 5.1 +/- 0.1). Mivazerol also showed low affinity for the [3H]clonidine-labelled I1 imidazoline receptors and for the [3H]idazoxan-labelled I2 receptors (pIC50 = 5.1 and 3.9, respectively). These results suggest that the non-adrenergic [3H]mivazerol binding sites are distinct from the imidazoline receptors in the human striatum.

  15. Binding of oligoarginine to membrane lipids and heparan sulfate: structural and thermodynamic characterization of a cell-penetrating peptide.

    PubMed

    Gonçalves, Elisabete; Kitas, Eric; Seelig, Joachim

    2005-02-22

    Cell-penetrating peptides (CPPs) comprise a group of arginine-rich oligopeptides that are able to deliver exogenous cargo into cells. A first step in the internalization of CPPs is their binding to the cell surface, a reaction likely to involve membrane phospholipids and/or heparan sulfate proteoglycans (HSPGs). The present work characterizes the interaction of R(9), one of the most efficient CPPs, with either heparan sulfate (HS) or lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). Isothermal titration calorimetry shows that R(9) binds to HS with high affinity. Assuming that HS has n independent and equivalent binding sites for R(9), we find an association constant of 3.1 x 10(6) M(-1) at 28 degrees C. At this temperature, the reaction enthalpy is DeltaH(degrees)pep = - 5.5 kcal/mol and approximately 7 R(9) molecules bind per HS chain, which is equivalent to approximately 0.95 cationic/anionic charge ratio. Delta decreases in magnitude upon an increase in temperature, and the reaction becomes entropy-driven at higher temperatures (>or=37 degrees C). The positive heat-capacity change entailed by this reaction (DeltaC(degrees)P = +167 cal mol(-1) K(-1)) indicates the loss of polar residues on R(9)-HS binding, suggesting that hydrophobic forces play no major role on binding. Calorimetric analysis of the interaction of R(9) with POPC/POPG (75:25) vesicles reveals an association constant of 8.2 x 10(4) M(-1) at 28 degrees C. Using a surface partition equilibrium model to correct for electrostatic effects, we find an intrinsic partition constant of approximately 900 M(-1), a value that is also confirmed by electrophoretic mobility measurements. This corresponds to an electrostatic contribution of approximately 33% to the total free energy of binding. Deuterium nuclear magnetic resonance (NMR) shows no change in the headgroup conformation of POPC and POPG, suggesting

  16. 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.

  17. 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.

  18. Increased affinity of histamine H1 binding to membranes of human myometrium at the end of pregnancy.

    PubMed

    Gonzalez, R; Echeverria, E; Reinicke, K; Rudolph, M I

    1994-12-01

    1. The characterization of H1 binding sites in membrane preparations of human myometrium obtained from pregnant and non-pregnant women was performed by using 3H-mepyramine as the radioactive ligand. 2. Saturation curve analysis revealed that 3H-mepyramine is bound to a single class of binding sites. Changes in the H1 site binding parameters were observed at the end of pregnancy, resulting in an increased affinity relative to non-pregnant tissue (Kd: 131.0 +/- 8.8 (non-pregnant) and 72.5 +/- 7.5 (pregnant) nM, n = 6, P < 0.01). 3. A reduction in receptor concentration at the end of pregnancy was also observed, [Bmax: 565.2 +/- 43.7 (non-pregnant) and 309.6 +/- 25.9 (pregnant) fmol/mg prot, n = 6, P < 0.01]. It is possible that this reduction in Bmax could be attributed to a dilution factor due to the increase in membraneous proteins that occurs during gestation.

  19. The role of apoproteins AI and AII in binding of high-density lipoprotein3 to membranes derived from bovine aortic endothelial cells.

    PubMed Central

    Vadiveloo, P K; Fidge, N H

    1992-01-01

    Although binding of high-density lipoproteins (HDL) to a variety of cells in culture has been widely reported, the mechanism of this binding has yet to be fully elucidated. The aim of the current studies was to explore the roles of apoproteins (apo) AI and AII in HDL3 binding to membranes derived from bovine aortic endothelial cells. Binding studies showed that HDL3 (which contains both apo AI and apo AII) and AII-HDL3 (which contain only apo AII) bound to membranes with similar affinity (44 +/- 6 and 41 +/- 9 micrograms/ml respectively) and capacity (673 +/- 97 and 969 +/- 101 ng bound/mg of membrane protein respectively). In contrast with these results, HDL3 [AI w/o AII] (which contain apo AI, but not apo AII) bound to the membranes with a significantly higher capacity (2228 +/- 206 ng bound/mg of membrane protein) and lower affinity (65 +/- 3 micrograms/ml) as compared with HDL3 or AII-HDL3. Therefore, although both apo AI and apo AII appear capable of facilitating HDL3 binding, the mechanisms involved probably differ. A model which fits the data postulates that a common receptor exists which binds both apo AI and apo AII, and that a particle containing AII can occupy up to four receptors (partly owing to each AII molecule containing two binding domains), whereas an HDL3 [AI w/o AII] particle can occupy only one. Images Fig. 3. PMID:1599393

  20. 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.

  1. A case of complete androgen insensitivity syndrome with a novel androgen receptor mutation.

    PubMed

    Chin, Vivian L; Sheffer-Babila, Sharone; Lee, Ting A; Tanaka, Kathryn; Zhou, Ping

    2012-01-01

    We report a case of a 14-year-old girl with primary amenorrhea and phenotypic as well as hormonal features of complete androgen insensitivity syndrome (CAIS), who tested positive for a novel missense androgen receptor gene mutation resulting in serine-to-isoleucine change at position 703 in exon 4 in the ligand-binding domain. The interesting features of this case include a persistence of Müllerian derivatives, Sertoli cell adenoma, Tanner III pubic hair, and a normal bone mineral density. These features are not typically described in CAIS. This novel mutation associated with a unique clinical presentation serves to significantly enrich the literature on this rare and fascinating disorder of androgen insensitivity syndrome.

  2. Inhibitors for Androgen Receptor Activation Surfaces

    DTIC Science & Technology

    2007-09-01

    new class of chemical therapeutics for treatment of prostate cancer. 15. SUBJECT TERMS X-ray crystallography, high throughput screening, medicinal... treatments because anti-androgen resis- tance usually develops. We conducted functional and x-ray screens to identify compounds that bind the AR surface and...possibility that such compounds could be used for prostate cancer treatment . It is unlikely that natural T3 or Triac concentrations approach levels required

  3. Effects of tricyclic compounds on membrane binding of bivalent cations, activities of acetylcholinesterase and some tissue proteases.

    PubMed

    Molnar, J; Sohar, I; Kovacs, J; Rakonczay, Z; Rausch, H

    1993-01-01

    A tricyclic compound tetrahydroaminoacridine is known to improve the cognitive function in Alzheimer's disease. The possible mechanism of action of acridine and structurally related tricyclic compounds was studied on the bivalent cation content of bacterial membrane, rat brain acetylcholinesterase and some tissue proteases in model experiments. Acridine orange and disubstituted chlorpromazine (CPZ) derivatives lowered Ca2+ and Mg2+ binding and membrane polarization in the simplest biological membrane (E. coli), as revealed by reactor neutron activation analysis. Acetylcholinesterase (AChE) was inhibited by CPZ, 3,7,8-trihydroxy-CPZ, acridine orange partially saturated desipramine, imipramine, trans-clopenthixol and tetrahydrocannabidiolic at 10(-4) to 10(-5). A metalloproteinase, MMP-7-ase, was inhibited by tetrahydrocannabidiolic acid, 3,7,8-trihydroxy-CPZ, acridine orange but other tissue proteinases, ATN-ase and cathepsin B, were less sensitive to these compounds. (ATN-ase is an acetyltyrosine-p-nitroanilide splitting enzyme, a serine protease). The chelate complex forming ability and electron donor capacity of the compounds may play a role in the biological effects tested. It is assumed that compounds which do not displace bivalent cations in membranes may exert an inhibitory effect on AChE, and that metalloproteinase enzymes may be promising for the treatment of degenerative brain diseases.

  4. Coordinated binding of Vps4 to ESCRT-III drives membrane neck constriction during MVB vesicle formation.

    PubMed

    Adell, Manuel Alonso Y; Vogel, Georg F; Pakdel, Mehrshad; Müller, Martin; Lindner, Herbert; Hess, Michael W; Teis, David

    2014-04-14

    Five endosomal sorting complexes required for transport (ESCRTs) mediate the degradation of ubiquitinated membrane proteins via multivesicular bodies (MVBs) in lysosomes. ESCRT-0, -I, and -II interact with cargo on endosomes. ESCRT-II also initiates the assembly of a ringlike ESCRT-III filament consisting of Vps20, Snf7, Vps24, and Vps2. The AAA-adenosine triphosphatase Vps4 disassembles and recycles the ESCRT-III complex, thereby terminating the ESCRT pathway. A mechanistic role for Vps4 in intraluminal vesicle (ILV) formation has been unclear. By combining yeast genetics, biochemistry, and electron tomography, we find that ESCRT-III assembly on endosomes is required to induce or stabilize the necks of growing MVB ILVs. Yet, ESCRT-III alone is not sufficient to complete ILV biogenesis. Rather, binding of Vps4 to ESCRT-III, coordinated by interactions with Vps2 and Snf7, is coupled to membrane neck constriction during ILV formation. Thus, Vps4 not only recycles ESCRT-III subunits but also cooperates with ESCRT-III to drive distinct membrane-remodeling steps, which lead to efficient membrane scission at the end of ILV biogenesis in vivo.

  5. Brevibacillin, a cationic lipopeptide that binds to lipoteichoic acid and subsequently disrupts cytoplasmic membrane of Staphylococcus aureus.

    PubMed

    Yang, Xu; Huang, En; Yousef, Ahmed E

    2017-01-01

    Brevibacillin is a newly-discovered antimicrobial lipopeptide produced by Brevibacillus laterosporus OSY-I1. It is active against Gram-positive bacteria, including antibiotic resistant strains. This research was initiated to investigate the mechanism of action of brevibacillin against an indicator strain, Staphylococcus aureus ATCC 6538. Results of the study proved that brevibacillin binds to lipoteichoic acid (LTA) on cell wall before interacting with cell membrane. Additionally, brevibacillin disrupts S. aureus cytoplasmic membrane by increasing its permeability, depolarization and potassium leakage. Therefore, cytoplasmic membrane serves as a major target for brevibacillin. Despite the presence of multiple sites on S. aureus cell envelope, scanning electron microscope observation didn't reveal evidence of cell lysis or any morphological defects in cells treated with brevibacillin. Based on the results of this study, we propose that the electrostatic interaction between the cationic brevibacillin and the anionic LTA helped the accumulation of the antimicrobial agent at cell surface; this was followed by translocation of the lipopeptide to the cytoplasmic membrane and disrupting its vital functions.

  6. Efficient protein immobilization on polyethersolfone electrospun nanofibrous membrane via covalent binding for biosensing applications.

    PubMed

    Mahmoudifard, Matin; Soudi, Sara; Soleimani, Masoud; Hosseinzadeh, Simzar; Esmaeili, Elaheh; Vossoughi, Manouchehr

    2016-01-01

    In this paper we introduce novel strategy for antibody immobilization using high surface area electrospun nanofibrous membrane based on ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling chemistry. To present the high performance of proposed biosensors, anti-staphylococcus enterotoxin B (anti-SEB) was used as a model to demonstrate the utility of our proposed system. Polymer solution of polyethersolfone was used to fabricate fine nanofibrous membrane. Moreover, industrial polyvinylidene fluoride membrane and conventional microtiter plate were also used to compare the efficiency of antibody immobilization. Scanning electron microscopy images were taken to study the morphology of the membranes. The surface activation of nanofibrous membrane was done with the help of O2 plasma. PES nanofibrous membrane with carboxyl functional groups for covalent attachment of antibodies were treated by EDC/NHS coupling agent. The quantity of antibody immobilization was measured by enzyme-linked immuno sorbent assay (ELISA) method. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy was performed to confirm the covalent immobilization of antibody on membrane. Atomic force microscopy, scanning electron microscopy and invert fluorescence microscopy were used to analyze the antibody distribution pattern on solid surfaces. Results show that oxygen plasma treatment effectively increased the amount of antibody immobilization through EDC/NHS coupling chemistry. It was found that the use of nanofibrous membrane causes the improved detection signal of ELISA based biosensors in comparison to the standard assay carried out in the 96-well microtiter plate. This method has the potential to improve the ELISA-based biosensor and we believe that this technique can be used in various biosensing methods.

  7. Structural basis for activation, assembly and membrane binding of ESCRT-III Snf7 filaments.

    PubMed

    Tang, Shaogeng; Henne, W Mike; Borbat, Peter P; Buchkovich, Nicholas J; Freed, Jack H; Mao, Yuxin; Fromme, J Christopher; Emr, Scott D

    2015-12-15

    The endosomal sorting complexes required for transport (ESCRTs) constitute hetero-oligomeric machines that catalyze multiple topologically similar membrane-remodeling processes. Although ESCRT-III subunits polymerize into spirals, how individual ESCRT-III subunits are activated and assembled together into a membrane-deforming filament remains unknown. Here, we determine X-ray crystal structures of the most abundant ESCRT-III subunit Snf7 in its active conformation. Using pulsed dipolar electron spin resonance spectroscopy (PDS), we show that Snf7 activation requires a prominent conformational rearrangement to expose protein-membrane and protein-protein interfaces. This promotes the assembly of Snf7 arrays with ~30 Å periodicity into a membrane-sculpting filament. Using a combination of biochemical and genetic approaches, both in vitro and in vivo, we demonstrate that mutations on these protein interfaces halt Snf7 assembly and block ESCRT function. The architecture of the activated and membrane-bound Snf7 polymer provides crucial insights into the spatially unique ESCRT-III-mediated membrane remodeling.

  8. Structure, function and binding selectivity and stereoselectivity of siderophore-iron outer membrane transporters.

    PubMed

    Schalk, Isabelle J; Mislin, Gaëtan L A; Brillet, Karl

    2012-01-01

    To get access to iron, microorganisms produce and release into their environment small organic metal chelators called siderophores. In parallel, they produce siderophore-iron outer membrane transporters (also called TonB-Dependent Transporters or TBDT) embedded in the outer membrane; these proteins actively reabsorb the siderophore loaded with iron from the extracellular medium. This active uptake requires energy in the form of the proton motive force transferred from the inner membrane to the outer membrane transporter via the inner membrane TonB complex. Siderophores produced by microorganisms are structurally very diverse with molecular weights of 150 up to 2000Da. Siderophore-iron uptake from the extracellular medium by TBDTs is a highly selective and sometimes even stereoselective process, with each siderophore having a specific TBDT. Unlike the siderophores, all TBDTs have similar structures and belong to the outer membrane β-barrel protein superfamily. The way in which the siderophore-iron complex passes through the TBDT is still unclear. In some bacteria, TBDTs are also partners of signaling cascades regulating the expression of proteins involved in siderophore biosynthesis and siderophore-iron acquisition.

  9. Structural basis for activation, assembly and membrane binding of ESCRT-III Snf7 filaments

    PubMed Central

    Tang, Shaogeng; Henne, W Mike; Borbat, Peter P; Buchkovich, Nicholas J; Freed, Jack H; Mao, Yuxin; Fromme, J Christopher; Emr, Scott D

    2015-01-01

    The endosomal sorting complexes required for transport (ESCRTs) constitute hetero-oligomeric machines that catalyze multiple topologically similar membrane-remodeling processes. Although ESCRT-III subunits polymerize into spirals, how individual ESCRT-III subunits are activated and assembled together into a membrane-deforming filament remains unknown. Here, we determine X-ray crystal structures of the most abundant ESCRT-III subunit Snf7 in its active conformation. Using pulsed dipolar electron spin resonance spectroscopy (PDS), we show that Snf7 activation requires a prominent conformational rearrangement to expose protein-membrane and protein-protein interfaces. This promotes the assembly of Snf7 arrays with ~30 Å periodicity into a membrane-sculpting filament. Using a combination of biochemical and genetic approaches, both in vitro and in vivo, we demonstrate that mutations on these protein interfaces halt Snf7 assembly and block ESCRT function. The architecture of the activated and membrane-bound Snf7 polymer provides crucial insights into the spatially unique ESCRT-III-mediated membrane remodeling. DOI: http://dx.doi.org/10.7554/eLife.12548.001 PMID:26670543

  10. Mutational analysis of the androgen receptor gene in two Chinese families with complete androgen insensitivity syndrome.

    PubMed

    Wang, Song; Xu, Haikun; An, Wei; Zhu, Dechun; Li, Dejun

    2016-06-01

    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.

  11. Prostasomes of canine seminal plasma - zinc-binding ability and effects on motility characteristics and plasma membrane integrity of spermatozoa.

    PubMed

    Mogielnicka-Brzozowska, M; Strzeżek, R; Wasilewska, K; Kordan, W

    2015-06-01

    Prostasomes are small lipid membrane-confined vesicles that are involved in various fertilization-related processes. The aim of this study was to demonstrate canine seminal plasma prostasomes' ability to bind zinc ions, as well as examining their effects on sperm motility characteristics and plasma membrane integrity during cold storage. Ejaculates, collected from five cross-bred dogs (n = 50), were subjected to ultracentrifugation followed by gel filtration (GF) on a Superose 6 column. Prostasomes appeared as a single fraction in the elution profile. Transmission electron microscopy (TEM) analysis of canine prostasomes revealed the presence of membrane vesicles with diameters ranging from 20.3 to 301 nm. The zinc-affinity chromatography on a Chelating Sepharose Fast Flow - Zn(2 +) showed that from 93 to 100% of the prostasome proteins bind zinc ions (P(+) Zn). SDS-PAGE revealed that canine P(+) Zn comprised four protein bands, with low molecular weights (10.2-12 kDa). We have also shown a positive effect of prostasomes (p < 0.05), especially variant B (2% of total seminal plasma protein) on canine sperm motility parameters after 2 h storage at 5°C (TMOT%, 44.75 ± 5.18) and PMOT%, 12.42 ± 1.59) and VAP, VSL, VCL, when compared with Control (TMOT%, 7.30 ± 1.41 and PMOT%, 1.70 ± 0.42). Higher percentage of spermatozoa with intact plasma membrane (SYBR/PI dual staining) and intact acrosome (Giemsa stained), after 2 h storage at 5°C, was showed, in variant A (1.5% of total seminal plasma protein) and B, when compared with Control and variant C (2.5% of total seminal plasma protein). The prostasomes' effect on motility and plasma membrane integrity of canine cold-stored spermatozoa may be related to their ability to bind zinc ions and regulate their availability to the sperm.

  12. Sulfated galactans from Gracilaria fisheri bind to shrimp haemocyte membrane proteins and stimulate the expression of immune genes.

    PubMed

    Rudtanatip, Tawut; Withyachumnarnkul, Boonsirm; Wongprasert, Kanokpan

    2015-11-01

    Previous studies demonstrated that sulfated galactans (SG) from Gracilaria fisheri (G. fisheri) exhibit immunostimulant activity in shrimp. The present study was conducted to test the hypothesis that SG stimulates signaling molecules of the immune response of shrimp by binding to receptors on the host cell membrane. Accordingly, we evaluated the ability of SG to bind to shrimp haemocytes and showed that SG bound to the shrimp haemocyte membrane (SHM), potentially to specific receptors. Furthermore, this binding was associated with an activation of immune response genes of shrimp. Data from confocal laser scanning micrographs revealed that FITC-labeled SG bound to haemocytes. Far western blot analysis demonstrated that SHM peptides, with molecular sizes of 13, 14, 15, 17, and 25 kDa, were associated with SG. Peptide sequence analysis of the isolated bands using LC-MS/MS and NCBI blast search revealed the identity of the 13, 14, and 17 kDa peptides as lipopolysaccharide and β-1,3-glucan binding protein (LGBP). SG induced the expression of immune related genes and downstream signaling mediators of LGBP including IMD, IKKs, NF-κB, antimicrobial peptides (crustin and PEN-4), the antiviral immunity (dicer), and proPO system (proPO-I and proPO-II). A LGBP neutralizing assay with anti-LGBP antibody indicated a decrease in SG-induced expression of LGBP downstream signaling mediators and the immune related genes. In conclusion, this study demonstrated that the SG-stimulated immune activity in haemocytes is mediated, in part, through the LGBP, and IMD-NF-κB pathway.

  13. Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

    SciTech Connect

    Walker, G.; Bourguignon, L.Y. )

    1990-08-01

    Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation.

  14. Comparison [(3)H]-flumazenil binding parameters in rat cortical membrane using different separation methods, filtration and centrifugation.

    PubMed

    Ahmadi, Fatemeh; Faizi, Mehrdad; Tabatabai, Sayyed Abbas; Beiki, Davood; Shahhosseini, Soraya

    2013-10-01

    Radioligand receptor binding assays are a common method to evaluate the affinity of newly synthesized benzodiazepine ligands for the receptor. [(3)H]-flumazenil is an antagonist of benzodiazepine receptors and is generally used as