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Sample records for intracellular channel protein

  1. Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na+ channels.

    Science.gov (United States)

    Hallaq, Haifa; Wang, Dao W; Kunic, Jennifer D; George, Alfred L; Wells, K Sam; Murray, Katherine T

    2012-02-01

    Na(+) current derived from expression of the cardiac isoform SCN5A is reduced by receptor-mediated or direct activation of protein kinase C (PKC). Previous work has suggested a possible role for loss of Na(+) channels at the plasma membrane in this effect, but the results are controversial. In this study, we tested the hypothesis that PKC activation acutely modulates the intracellular distribution of SCN5A channels and that this effect can be visualized in living cells. In human embryonic kidney cells that stably expressed SCN5A with green fluorescent protein (GFP) fused to the channel COOH-terminus (SCN5A-GFP), Na(+) currents were suppressed by an exposure to PKC activation. Using confocal microscopy, colocalization of SCN5A-GFP channels with the plasma membrane under control and stimulated conditions was quantified. A separate population of SCN5A channels containing an extracellular epitope was immunolabeled to permit temporally stable labeling of the plasma membrane. Our results demonstrated that Na(+) channels were preferentially trafficked away from the plasma membrane by PKC activation, with a major contribution by Ca(2+)-sensitive or conventional PKC isoforms, whereas stimulation of protein kinase A (PKA) had the opposite effect. Removal of the conserved PKC site Ser(1503) or exposure to the NADPH oxidase inhibitor apocynin eliminated the PKC-mediated effect to alter channel trafficking, indicating that both channel phosphorylation and ROS were required. Experiments using fluorescence recovery after photobleaching demonstrated that both PKC and PKA also modified channel mobility in a manner consistent with the dynamics of channel distribution. These results demonstrate that the activation of protein kinases can acutely regulate the intracellular distribution and molecular mobility of cardiac Na(+) channels in living cells.

  2. Bioinformatic Characterization of the Trimeric Intracellular Cation-Specific Channel Protein Family

    Science.gov (United States)

    Silverio, Abe L. F.

    2014-01-01

    Trimeric intracellular cation-specific (TRIC) channels are integral to muscle excitation–contraction coupling. TRIC channels provide counter-ionic flux when calcium is rapidly transported from intracellular stores to the cell cytoplasm. Until recently, knowledge of the presence of these proteins was limited to animals. We analyzed the TRIC family and identified a profusion of prokaryotic family members with topologies and motifs similar to those of their eukaryotic counterparts. Prokaryotic members far outnumber eukaryotic members, and although none has been functionally characterized, the evidence suggests that they function as secondary carriers. The presence of fused N- or C-terminal domains of known biochemical functions as well as genomic context analyses provide clues about the functions of these prokaryotic homologs. They are proposed to function in metabolite (e.g., amino acid/ nucleotide) efflux. Phylogenetic analysis revealed that TRIC channel homologs diverged relatively early during evolutionary history and that horizontal gene transfer was frequent in prokaryotes but not in eukaryotes. Topological analyses of TRIC channels revealed that these proteins possess seven putative transmembrane segments (TMSs), which arose by intragenic duplication of a three-TMS polypeptide-encoding genetic element followed by addition of a seventh TMS at the C terminus to give the precursor of all current TRIC family homologs. We propose that this family arose in prokaryotes. PMID:21519847

  3. Regulation of the membrane insertion and conductance activity of the metamorphic chloride intracellular channel protein CLIC1 by cholesterol.

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    Stella M Valenzuela

    Full Text Available The Chloride Intracellular ion channel protein CLIC1 has the ability to spontaneously insert into lipid membranes from a soluble, globular state. The precise mechanism of how this occurs and what regulates this insertion is still largely unknown, although factors such as pH and redox environment are known contributors. In the current study, we demonstrate that the presence and concentration of cholesterol in the membrane regulates the spontaneous insertion of CLIC1 into the membrane as well as its ion channel activity. The study employed pressure versus area change measurements of Langmuir lipid monolayer films; and impedance spectroscopy measurements using tethered bilayer membranes to monitor membrane conductance during and following the addition of CLIC1 protein. The observed cholesterol dependent behaviour of CLIC1 is highly reminiscent of the cholesterol-dependent-cytolysin family of bacterial pore-forming proteins, suggesting common regulatory mechanisms for spontaneous protein insertion into the membrane bilayer.

  4. Intracellular ion channels and cancer

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    Luigi eLeanza

    2013-09-01

    Full Text Available Several types of channels play a role in the maintenance of ion homeostasis in subcellular organelles including endoplasmatic reticulum, nucleus, lysosome, endosome and mitochondria. Here we give a brief overview of the contribution of various mitochondrial and other organellar channels to cancer cell proliferation or death. Much attention is focused on channels involved in intracellular calcium signaling and on ion fluxes in the ATP-producing organelle mitochondria. Mitochondrial K+ channels (Ca2+-dependent BKCa and IKCa, ATP-dependent KATP, Kv1.3, two-pore TWIK-related Acid-Sensitive K+ channel-3 (TASK-3, Ca2+ uniporter MCU, Mg2+-permeable Mrs2, anion channels (voltage-dependent chloride channel VDAC, intracellular chloride channel CLIC and the Permeability Transition Pore (MPTP contribute importantly to the regulation of function in this organelle. Since mitochondria play a central role in apoptosis, modulation of their ion channels by pharmacological means may lead to death of cancer cells. The nuclear potassium channel Kv10.1 and the nuclear chloride channel CLIC4 as well as the endoplasmatic reticulum (ER-located inositol 1,4,5-trisphosphate (IP3 receptor, the ER-located Ca2+ depletion sensor STIM1 (stromal interaction molecule 1, a component of the store-operated Ca2+ channel and the ER-resident TRPM8 are also mentioned. Furthermore, pharmacological tools affecting organellar channels and modulating cancer cell survival are discussed. The channels described in this review are summarized on Figure 1. Overall, the view is emerging that intracellular ion channels may represent a promising target for cancer treatment.

  5. An intracellular anion channel critical for pigmentation.

    Science.gov (United States)

    Bellono, Nicholas W; Escobar, Iliana E; Lefkovith, Ariel J; Marks, Michael S; Oancea, Elena

    2014-12-16

    Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation.

  6. Structural dynamics of soluble chloride intracellular channel protein CLIC1 examined by amide hydrogen-deuterium exchange mass spectrometry.

    Science.gov (United States)

    Stoychev, Stoyan H; Nathaniel, Christos; Fanucchi, Sylvia; Brock, Melissa; Li, Sheng; Asmus, Kyle; Woods, Virgil L; Dirr, Heini W

    2009-09-08

    Chloride intracellular channel protein 1 (CLIC1) functions as an anion channel in plasma and nuclear membranes when its soluble monomeric form converts to an integral-membrane form. The transmembrane region of CLIC1 is located in its thioredoxin-like domain 1, but the mechanism whereby the protein converts to its membrane conformation has yet to be determined. Since channel formation in membranes is enhanced at low pH (5 to 5.5), a condition that is found at the surface of membranes, the structural dynamics of soluble CLIC1 was studied at pH 7 and at pH 5.5 in the absence of membranes by amide hydrogen-deuterium exchange mass spectrometry (DXMS). Rapid hydrogen exchange data indicate that CLIC1 displays a similar core structure at these pH values. Domain 1 is less stable than the all-helical domain 2, and, while the structure of domain 1 remains intact, its conformational flexibility is further increased in an acidic environment (pH 5.5). In the absence of membrane, an acidic environment appears to prime the solution structure of CLIC1 by destabilizing domain 1 in order to lower the activation energy barrier for its conversion to the membrane-insertion conformation. The significantly enhanced H/D-exchange rates at pH 5.5 displayed by two segments (peptides 11-31 and 68-82) could be due to the protonation of acidic residues in salt bridges. One of these segments (peptide 11-31) includes part of the transmembrane region which, in the solution structure, consists of helix alpha1. This helix is intrinsically stable and is most likely retained in the membrane conformation. Strand beta2, another element of the transmembrane region, displays a propensity to form a helical structure and has putative N- and C-capping motifs, suggesting that it too most likely forms a helix in a lipid bilayer.

  7. An intracellular anion channel critical for pigmentation

    Science.gov (United States)

    Bellono, Nicholas W; Escobar, Iliana E; Lefkovith, Ariel J; Marks, Michael S; Oancea, Elena

    2014-01-01

    Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation. DOI: http://dx.doi.org/10.7554/eLife.04543.001 PMID:25513726

  8. Intracellular protein breakdown. 8

    International Nuclear Information System (INIS)

    Bohley, P.; Kirschke, H.; Langner, J.; Wiederanders, B.; Ansorge, S.

    1976-01-01

    Double-labelled proteins from rat liver cytosol ( 14 C in long-lived, 3 H in short-lived proteins after in-vivo-labelling) are used as substrates for unlabelled proteinases in vitro. Differences in the degradation rates of short-lived and long-lived proteins in vitro by different proteinases and after addition of different effectors allow conclusions concerning their importance for the in-vivo-turnover of substrate proteins. The main activity (>90%) of soluble lysosomal proteinases at pH 6.1 and pH 6.9 is caused by thiolproteinases, which degrade preferentially short-lived cytosol proteins. These proteinases are inhibited by leupeptin. Autolysis of double-labelled cell fractions shows a remarkably faster breakdown of short-lived substrate proteins only in the soluble part of lysosomes. Microsomal fractions degrade in vitro preferentially long-lived substrate proteins. (author)

  9. Identification of Potent Chloride Intracellular Channel Protein 1 Inhibitors from Traditional Chinese Medicine through Structure-Based Virtual Screening and Molecular Dynamics Analysis

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    Wei Wang

    2017-01-01

    Full Text Available Chloride intracellular channel 1 (CLIC1 is involved in the development of most aggressive human tumors, including gastric, colon, lung, liver, and glioblastoma cancers. It has become an attractive new therapeutic target for several types of cancer. In this work, we aim to identify natural products as potent CLIC1 inhibitors from Traditional Chinese Medicine (TCM database using structure-based virtual screening and molecular dynamics (MD simulation. First, structure-based docking was employed to screen the refined TCM database and the top 500 TCM compounds were obtained and reranked by X-Score. Then, 30 potent hits were achieved from the top 500 TCM compounds using cluster and ligand-protein interaction analysis. Finally, MD simulation was employed to validate the stability of interactions between each hit and CLIC1 protein from docking simulation, and Molecular Mechanics/Generalized Born Surface Area (MM-GBSA analysis was used to refine the virtual hits. Six TCM compounds with top MM-GBSA scores and ideal-binding models were confirmed as the final hits. Our study provides information about the interaction between TCM compounds and CLIC1 protein, which may be helpful for further experimental investigations. In addition, the top 6 natural products structural scaffolds could serve as building blocks in designing drug-like molecules for CLIC1 inhibition.

  10. Structural Dynamics of Soluble Chloride Intracellular Channel Protein CLIC1 Examined by Amide Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS)†

    Science.gov (United States)

    Stoychev, Stoyan H.; Nathaniel, Christos; Fanucchi, Sylvia; Brock, Melissa; Li, Sheng; Asmus, Kyle; Woods, Virgil L.; Dirr, Heini W.

    2009-01-01

    Chloride intracellular channel protein 1 (CLIC1) functions as an anion channel in plasma and nuclear membranes when its soluble monomeric form converts to an integral-membrane form. The transmembrane region of CLIC1 is located in its thioredoxin-like domain 1 but the mechanism whereby the protein converts to its membrane conformation has yet to be determined. Since channel formation in membranes is enhanced at low pH (5 to 5.5), a condition that is found at the surface of membranes, the structural dynamics of soluble CLIC1 was studied at pH 7 and at pH 5.5 in the absence of membranes by amide hydrogen-deuterium exchange mass spectrometry (DXMS). Rapid hydrogen exchange data indicate that CLIC1 displays a similar core structure at these pH values. Domain 1 is less stable than the all-helical domain 2 and, while the structure of domain 1 remains intact, its conformational flexibility is further increased in an acidic environment (pH 5.5). In the absence of membrane, an acidic environment appears to prime the solution structure of CLIC1 by destabilising domain 1 in order to lower the activation energy barrier for its conversion to the membrane-insertion conformation. The significantly enhanced H/D-exchange rates at pH 5.5 displayed by two segments (peptides 11-31 and 68-82) could be due to the protonation of acidic residues in salt bridges. One of these segments (peptide 11-31) includes part of the transmembrane region which, in the solution structure, consists of helix α1. This helix is intrinsically stable and is most likely retained in the membrane conformation. Strand β2, another element of the transmembrane region, displays a propensity to form a helical structure and has putative N- and C-capping motifs, suggesting that it too most likely forms a helix in a lipid bilayer. PMID:19650640

  11. Crystal structures of the TRIC trimeric intracellular cation channel orthologues.

    Science.gov (United States)

    Kasuya, Go; Hiraizumi, Masahiro; Maturana, Andrés D; Kumazaki, Kaoru; Fujiwara, Yuichiro; Liu, Keihong; Nakada-Nakura, Yoshiko; Iwata, So; Tsukada, Keisuke; Komori, Tomotaka; Uemura, Sotaro; Goto, Yuhei; Nakane, Takanori; Takemoto, Mizuki; Kato, Hideaki E; Yamashita, Keitaro; Wada, Miki; Ito, Koichi; Ishitani, Ryuichiro; Hattori, Motoyuki; Nureki, Osamu

    2016-12-01

    Ca 2+ release from the sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) is crucial for muscle contraction, cell growth, apoptosis, learning and memory. The trimeric intracellular cation (TRIC) channels were recently identified as cation channels balancing the SR and ER membrane potentials, and are implicated in Ca 2+ signaling and homeostasis. Here we present the crystal structures of prokaryotic TRIC channels in the closed state and structure-based functional analyses of prokaryotic and eukaryotic TRIC channels. Each trimer subunit consists of seven transmembrane (TM) helices with two inverted repeated regions. The electrophysiological, biochemical and biophysical analyses revealed that TRIC channels possess an ion-conducting pore within each subunit, and that the trimer formation contributes to the stability of the protein. The symmetrically related TM2 and TM5 helices are kinked at the conserved glycine clusters, and these kinks are important for the channel activity. Furthermore, the kinks of the TM2 and TM5 helices generate lateral fenestrations at each subunit interface. Unexpectedly, these lateral fenestrations are occupied with lipid molecules. This study provides the structural and functional framework for the molecular mechanism of this ion channel superfamily.

  12. Efficient intracellular delivery of native proteins

    NARCIS (Netherlands)

    D'Astolfo, Diego S; Pagliero, Romina J; Pras, Anita; Karthaus, Wouter R; Clevers, Hans; Prasad, Vikram; Lebbink, Robert Jan; Rehmann, Holger; Geijsen, Niels

    2015-01-01

    Modulation of protein function is used to intervene in cellular processes but is often done indirectly by means of introducing DNA or mRNA encoding the effector protein. Thus far, direct intracellular delivery of proteins has remained challenging. We developed a method termed iTOP, for induced

  13. Intracellular Protein Delivery for Treating Breast Cancer

    Science.gov (United States)

    2014-08-01

    Intracellular delivery of such proteins, including human tumor suppressors (such as p53) (Brown et al., 2009) and exogenous tumor-killing proteins...vivo systems. Nature materials 11, 1038-1043. Chorny, M., Hood, E., Levy, R.J., and Muzykantov, V.R. (2010). Endothelial delivery of antioxidant ...for the ntracellular delivery of such proteins, including human umor suppressors [7] and exogenous tumor-killing proteins 8—10]), is attractive as a

  14. Calcium, channels, intracellular signaling and autoimmunity.

    Science.gov (United States)

    Izquierdo, Jorge-Hernán; Bonilla-Abadía, Fabio; Cañas, Carlos A; Tobón, Gabriel J

    2014-01-01

    Calcium (Ca²⁺) is an important cation able to function as a second messenger in different cells of the immune system, particularly in B and T lymphocytes, macrophages and mastocytes, among others. Recent discoveries related to the entry of Ca²⁺ through the store-operated calcium entry (SOCE) has opened a new investigation area about the cell destiny regulated by Ca²⁺ especially in B and T lymphocytes. SOCE acts through calcium-release-activated calcium (CRAC) channels. The function of CRAC depends of two recently discovered regulators: the Ca²⁺ sensor in the endoplasmic reticulum or stromal interaction molecule (STIM-1) and one subunit of CRAC channels called Orai1. This review focuses on the role of Ca²⁺ signals in B and T lymphocytes functions, the signalling pathways leading to Ca²⁺ influx, and the relationship between Ca²⁺ signals and autoimmune diseases. Copyright © 2013 Elsevier España, S.L. All rights reserved.

  15. CONTRIBUTIONS OF INTRACELLULAR IONS TO Kv CHANNEL VOLTAGE SENSOR DYNAMICS.

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    Samuel eGoodchild

    2012-06-01

    Full Text Available Voltage sensing domains of Kv channels control ionic conductance through coupling of the movement of charged residues in the S4 segment to conformational changes at the cytoplasmic region of the pore domain, that allow K+ ions to flow. Conformational transitions within the voltage sensing domain caused by changes in the applied voltage across the membrane field are coupled to the conducting pore region and the gating of ionic conductance. However, several other factors not directly linked to the voltage dependent movement of charged residues within the voltage sensor impact the dynamics of the voltage sensor, such as inactivation, ionic conductance, intracellular ion identity and block of the channel by intracellular ligands. The effect of intracellular ions on voltage sensor dynamics is of importance in the interpretation of gating current measurements and the physiology of pore/voltage sensor coupling. There is a significant amount of variability in the reported kinetics of voltage sensor deactivation kinetics of Kv channels attributed to different mechanisms such as open state stabilization, immobilization and relaxation processes of the voltage sensor. Here we separate these factors and focus on the causal role that intracellular ions can play in allosterically modulating the dynamics of Kv voltage sensor deactivation kinetics. These considerations are of critical importance in understanding the molecular determinants of the complete channel gating cycle from activation to deactivation.

  16. Temporal protein expression pattern in intracellular signalling ...

    Indian Academy of Sciences (India)

    2015-09-28

    Sep 28, 2015 ... [Ganguli P, Chowdhury S, Bhowmick R and Sarkar RR 2015 Temporal protein expression pattern in intracellular signalling cascade during T-cell activation: A ... cells and tissues by studying different signalling pathways, such as Hedgehog ...... Murray JD 2003 On the mechanochemical theory of biological.

  17. The transduction channel TRPM5 is gated by intracellular calcium in taste cells.

    Science.gov (United States)

    Zhang, Zheng; Zhao, Zhen; Margolskee, Robert; Liman, Emily

    2007-05-23

    Bitter, sweet, and umami tastants are detected by G-protein-coupled receptors that signal through a common second-messenger cascade involving gustducin, phospholipase C beta2, and the transient receptor potential M5 (TRPM5) ion channel. The mechanism by which phosphoinositide signaling activates TRPM5 has been studied in heterologous cell types with contradictory results. To resolve this issue and understand the role of TRPM5 in taste signaling, we took advantage of mice in which the TRPM5 promoter drives expression of green fluorescent protein and mice that carry a targeted deletion of the TRPM5 gene to unequivocally identify TRPM5-dependent currents in taste receptor cells. Our results show that brief elevation of intracellular inositol trisphosphate or Ca2+ is sufficient to gate TRPM5-dependent currents in intact taste cells, but only intracellular Ca2+ is able to activate TRPM5-dependent currents in excised patches. Detailed study in excised patches showed that TRPM5 forms a nonselective cation channel that is half-activated by 8 microM Ca2+ and that desensitizes in response to prolonged exposure to intracellular Ca2+. In addition to channels encoded by the TRPM5 gene, we found that taste cells have a second type of Ca2+-activated nonselective cation channel that is less sensitive to intracellular Ca2+. These data constrain proposed models for taste transduction and suggest a link between receptor signaling and membrane potential in taste cells.

  18. Intracellular Ca2+ and the phospholipid PIP2 regulate the taste transduction ion channel TRPM5.

    Science.gov (United States)

    Liu, Dan; Liman, Emily R

    2003-12-09

    The transduction of taste is a fundamental process that allows animals to discriminate nutritious from noxious substances. Three taste modalities, bitter, sweet, and amino acid, are mediated by G protein-coupled receptors that signal through a common transduction cascade: activation of phospholipase C beta2, leading to a breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol and inositol 1,4,5-trisphosphate, which causes release of Ca2+ from intracellular stores. The ion channel, TRPM5, is an essential component of this cascade; however, the mechanism by which it is activated is not known. Here we show that heterologously expressed TRPM5 forms a cation channel that is directly activated by micromolar concentrations of intracellular Ca2+ (K1/2 = 21 microM). Sustained exposure to Ca2+ desensitizes TRPM5 channels, but PIP2 reverses desensitization, partially restoring channel activity. Whole-cell TRPM5 currents can be activated by intracellular Ca2+ and show strong outward rectification because of voltage-sensitive gating of the channels. TRPM5 channels are nonselective among monovalent cations and not detectably permeable to divalent cations. We propose that the regulation of TRPM5 by Ca2+ mediates sensory activation in the taste system.

  19. Ca2+ Binding/Permeation via Calcium Channel, CaV1.1, Regulates the Intracellular Distribution of the Fatty Acid Transport Protein, CD36, and Fatty Acid Metabolism.

    Science.gov (United States)

    Georgiou, Dimitra K; Dagnino-Acosta, Adan; Lee, Chang Seok; Griffin, Deric M; Wang, Hui; Lagor, William R; Pautler, Robia G; Dirksen, Robert T; Hamilton, Susan L

    2015-09-25

    Ca(2+) permeation and/or binding to the skeletal muscle L-type Ca(2+) channel (CaV1.1) facilitates activation of Ca(2+)/calmodulin kinase type II (CaMKII) and Ca(2+) store refilling to reduce muscle fatigue and atrophy (Lee, C. S., Dagnino-Acosta, A., Yarotskyy, V., Hanna, A., Lyfenko, A., Knoblauch, M., Georgiou, D. K., Poché, R. A., Swank, M. W., Long, C., Ismailov, I. I., Lanner, J., Tran, T., Dong, K., Rodney, G. G., Dickinson, M. E., Beeton, C., Zhang, P., Dirksen, R. T., and Hamilton, S. L. (2015) Skelet. Muscle 5, 4). Mice with a mutation (E1014K) in the Cacna1s (α1 subunit of CaV1.1) gene that abolishes Ca(2+) binding within the CaV1.1 pore gain more body weight and fat on a chow diet than control mice, without changes in food intake or activity, suggesting that CaV1.1-mediated CaMKII activation impacts muscle energy expenditure. We delineate a pathway (Cav1.1→ CaMKII→ NOS) in normal skeletal muscle that regulates the intracellular distribution of the fatty acid transport protein, CD36, altering fatty acid metabolism. The consequences of blocking this pathway are decreased mitochondrial β-oxidation and decreased energy expenditure. This study delineates a previously uncharacterized CaV1.1-mediated pathway that regulates energy utilization in skeletal muscle. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Functional conservation study of polarity protein Crumbs intracellular domain.

    Science.gov (United States)

    Shi, Qi-ping; Cao, Hao-wei; Xu, Rui; Zhang, Dan-dan; Huang, Juan

    2017-01-20

    The transmembrane protein Crumbs (Crb) plays key roles in the establishing and maintaining cell apical-basal polarity in epithelial cells by determining the apical plasma membrane identity. Although its intracellular domain contains only 37 amino acids, it is absolutely essential for its function. In Drosophila, mutations in this intracellular domain result in severe defects in epithelial polarity and abnormal embryonic development. The intracellular domain of Crb shows high homology across species from Drosophila to Mus musculus and Homo sapiens. However, the intracellular domains of the two Crb proteins in C. elegans are rather divergent from those of Drosophila and mammals, raising the question on whether the function of the intracellular domain of the Crb protein is conserved in C. elegans. Using genomic engineering approach, we replaced the intracellular domain of the Drosophila Crb with that of C. elegans Crb2 (CeCrb2), which has extremely low homology with those from the Crb proteins of Drosophila and mammals. Surprisingly, substituting the intracellular domain of Drosophila Crb with that of CeCrb2 did not cause any abnormalities in development of the Drosophila embryo, in terms of expression and localization of Crb and other polarity proteins and apical-basal polarity in embryonic epithelial cells. Our results support the notion that despite their extensive sequence variations, all functionally critical amino acid residues and motifs of the intercellular domain of Crb proteins are fully conserved between Drosophila and C. elegans.

  1. Immune regulation of Rab proteins expression and intracellular transport.

    Science.gov (United States)

    Pei, Gang; Bronietzki, Marc; Gutierrez, Maximiliano Gabriel

    2012-07-01

    Compartmentalization in cells of the immune system, the focus of this review, facilitates the spatiotemporal organization of cellular responses essential for specialized immune functions. In this process of compartment maintenance, Rab proteins are central regulators of protein-mediated transport and fusion of intracellular structures. It is widely believed that the intracellular concentration of proteins that regulate intracellular transport, including Rab proteins, is constitutively mantained. However, there is a growing body of evidence indicating that transcriptional rates of Rab proteins can be modified. This process is especially evident during immune activation and argues that after activation, these cells require higher levels of Rab proteins. The aim of this review is to discuss evidence showing the increasing links between Rab protein expression and intracellular transport, particularly in monocytes and macrophages. We highlight here biological processes in which the expression of Rab GTPases is selectively regulated, leading to the activation of specific intracellular routes. Further, we focus on the immune regulation of intracellular transport after cytokine activation and microbial infection, with an emphasis in mycobacterial infection.

  2. Intracellular long-chain acyl CoAs activate TRPV1 channels.

    Directory of Open Access Journals (Sweden)

    Yi Yu

    Full Text Available TRPV1 channels are an important class of membrane proteins that play an integral role in the regulation of intracellular cations such as calcium in many different tissue types. The anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2 is a known positive modulator of TRPV1 channels and the negatively charged phosphate groups interact with several basic amino acid residues in the proximal C-terminal TRP domain of the TRPV1 channel. We and other groups have shown that physiological sub-micromolar levels of long-chain acyl CoAs (LC-CoAs, another ubiquitous anionic lipid, can also act as positive modulators of ion channels and exchangers. Therefore, we investigated whether TRPV1 channel activity is similarly regulated by LC-CoAs. Our results show that LC-CoAs are potent activators of the TRPV1 channel and interact with the same PIP2-binding residues in TRPV1. In contrast to PIP2, LC-CoA modulation of TRPV1 is independent of Ca2+i, acting in an acyl side-chain saturation and chain-length dependent manner. Elevation of LC-CoAs in intact Jurkat T-cells leads to significant increases in agonist-induced Ca2+i levels. Our novel findings indicate that LC-CoAs represent a new fundamental mechanism for regulation of TRPV1 channel activity that may play a role in diverse cell types under physiological and pathophysiological conditions that alter fatty acid transport and metabolism such as obesity and diabetes.

  3. Cytoplasmic tail of coronavirus spike protein has intracellular ...

    Indian Academy of Sciences (India)

    2017-04-18

    Apr 18, 2017 ... if the histidine residue is protonated. Lontok et al., in their chimeric S protein studies used C terminal 11 amino acids of SARS-S protein attached to the plasma membrane re- porter protein VSV-G to show KXHXX motif is an intra- cellular localization signal for SARS, and the intracellular distribution closely ...

  4. Temporal protein expression pattern in intracellular signalling ...

    Indian Academy of Sciences (India)

    2015-09-28

    searched using PUBMED and. Google Scholar). The protein-protein interaction data obtain- ed were knitted together to reconstruct the entire signalling cascade. The diagram of the reconstructed pathway was drawn using ...

  5. Temporal protein expression pattern in intracellular signalling ...

    Indian Academy of Sciences (India)

    2016-08-26

    Aug 26, 2016 ... Various T-cell co-receptor molecules and calcium channel CRAC play a pivotal role in the maintenance of cell's functional responses by regulating the production of effector molecules (mostly cytokines) that aids in immune clearance and also maintaining the cell in a functionally active state. Any defect in ...

  6. Analysis of intracellular expressed proteins of Mycobacterium tuberculosis clinical isolates

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    Singhal Neelja

    2012-03-01

    Full Text Available Abstract Background Tuberculosis (TB is the most threatening infectious disease globally. Although progress has been made to reduce global incidence of TB, emergence of multidrug resistant (MDR TB threatens to undermine these advances. To combat the disease, novel intervention strategies effective against drug resistant and sensitive subpopulations of M. tuberculosis are urgently required as adducts in the present treatment regimen. Using THP-1 cells we have analyzed and compared the global protein expression profile of broth-cultured and intraphagosomally grown drug resistant and sensitive M.tuberculosis clinical isolates. Results On comparing the two dimensional (2-DE gels, many proteins were found to be upregulated/expressed during intracellular state which were identified by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS. Four proteins (adenosylhomocysteinase, aspartate carbomyltransferase, putatitive thiosulfate sulfurtransferase and universal stress protein were present in both intracellular MDR and sensitive isolates and three of these belonged to intermediary metabolism and respiration category. Two proteins (alanine dehydrogenase and adenosine kinase of intracellular MDR isolate and two (glucose-6-phosphate isomerase and ATP synthase epsilon chain of intracellular sensitive isolate belonged to intermediary metabolism and respiration category. One protein (Peroxidase/Catalase of intracellular MDR and three (HSPX, 14 kDa antigen and 10 kDa chaperonin of sensitive isolate belonged to virulence, detoxification and adaptation category. ESAT-6 of intracellular MDR belonged to cell wall and cell processes category. Two proteins (Antigen 85-C and Antigen 85-A of intracellular sensitive isolate were involved in lipid metabolism while probable peptidyl-prolyl cis-trans isomerase A was involved in information pathways. Four (Rv0635, Rv1827, Rv0036c and Rv2032 of intracellular MDR and two proteins (Rv2896c and Rv2558c of

  7. Cytoplasmic tail of coronavirus spike protein has intracellular

    Indian Academy of Sciences (India)

    Intracellular trafficking and localization studies of spike protein from SARS and OC43 showed that SARS spikeprotein is localized in the ER or ERGIC compartment and OC43 spike protein is predominantly localized in thelysosome. Differential localization can be explained by signal sequence. The sequence alignment ...

  8. Confocal microscopy for intracellular co-localization of proteins.

    Science.gov (United States)

    Miyashita, Toshiyuki

    2015-01-01

    Confocal laser scanning microscopy is the best method to visualize intracellular co-localization of proteins in intact cells. Because of the point scan/pinhole detection system, light contribution from the neighborhood of the scanning spot in the specimen can be eliminated, allowing high Z-axis resolution. Fluorescence detection by sensitive photomultiplier tubes allows the usage of filters with a narrow bandpath, resulting in minimal cross-talk (overlap) between two spectra. This is particularly important in demonstrating co-localization of proteins with multicolor labeling. Here, the methods outlining the detection of transiently expressed tagged proteins and the detection of endogenous proteins are described. Ideally, the intracellular co-localization of two endogenous proteins should be demonstrated. However, when antibodies raised against the protein of interest are unavailable for immunofluorescence or the available cell lines do not express the protein of interest sufficiently enough for immunofluorescence, an alternative method is to transfect cells with expression plasmids that encode tagged proteins and stain the cells with anti-tag antibodies. However, it should be noted that the tagging of proteins of interest or their overexpression could potentially alter the intracellular localization or the function of the target protein.

  9. Molecular evolution, intracellular organization, and the quinary structure of proteins.

    OpenAIRE

    McConkey, E H

    1982-01-01

    High-resolution two-dimensional polyacrylamide gel electrophoresis shows that at least half of 370 denatured polypeptides from hamster cells and human cells are indistinguishable in terms of isoelectric points and molecular weights. Molecular evolution may have been more conservative for this set of proteins than sequence studies on soluble proteins have implied. This may be a consequence of complexities of intracellular organization and the numerous macromolecular interactions in which most ...

  10. Intracellular Na+ inhibits volume regulated anion channel in rat cortical astrocytes

    Czech Academy of Sciences Publication Activity Database

    Minieri, L.; Pivoňková, Helena; Harantová, Lenka; Anděrová, Miroslava; Ferroni, S.

    2015-01-01

    Roč. 132, č. 3 (2015), s. 286-300 ISSN 0022-3042 R&D Projects: GA ČR(CZ) GBP304/12/G069; GA ČR GA13-02154S Institutional support: RVO:68378041 Keywords : volume-regulated anion channels * intracellular sodium * cell volume Subject RIV: FH - Neurology Impact factor: 3.842, year: 2015

  11. Intracellular transport proteins: classification, structure and function of kinesins

    Directory of Open Access Journals (Sweden)

    Agnieszka Chudy

    2011-09-01

    Full Text Available Correct cell functioning, division and morphogenesis rely on efficient intracellular transport. Apart from dyneins and myosins, kinesins are the main proteins responsible for intracellular movement. Kinesins are a large, diverse group of motor proteins, which based on phylogenetic similarity were classified into fourteen families. Among these families, due to the location of their motor domains, three groups have been characterized: N-, C- and M-kinesin. As molecular motors, kinesins transport various molecules and vesicles mainly towards the microtubule plus end (from the cell body participating in anterograde transport, although there are also kinesins involved in retrograde transport (C-kinesins. Kinesins are also involved in spindle formation, chromosome segregation, and spermatogenesis. Because of their great importance for the correct functioning of cells, mutations in kinesin coding genes may lead to such neurodegenerative diseases as dominant hereditary spastic paraplegia or Charcot-Marie-Tooth disease.

  12. Bioreducible Lipid-like Nanoparticles for Intracellular Protein Delivery

    Science.gov (United States)

    Arellano, Carlos Luis

    Protein-based therapy is one of the most direct ways to manipulate cell function and treat human disease. Although protein therapeutics has made its way to clinical practice, with five of the top fifteen global pharmaceuticals being peptide or protein-based drugs, one common limitation is that the effects of protein therapy are only achieved through the targeting of cell surface receptors and intracellular domains. Due to the impermeability of the cell membrane to most foreign materials, entire classes of potentially therapeutic proteins cannot thoroughly be studied without a safe and efficient method of transporting proteins into the cytosol. We report the use of a combinatorially-designed bioreducible lipid-like material (termed "lipidoid") - based protein delivery platform for the transfection of human cancer cell lines. Lipidoid nanoparticles are synthesized through a thin film dispersion method. The degradation of the bioreducible nanoparticles was observed when exposed to glutathione, a highly reductive compound present in the cytosol. We demonstrate that the nanoparticles are capable of transfecting a dose-dependent concentration of our model protein, beta-galactosidase into HeLa cells. Furthermore, formulations of the lipidoid containing the cytotoxic proteins saporin and RNase-A are both capable of inhibiting tumor cell proliferation as observed in in vitro treatment of different human cancer cell lines. There was no observed loss in protein activity after lyophilization and long--term storage, indicating the potential of pre-clinical applications. Overall, we demonstrate an effective approach to protein formulation and intracellular delivery. We believe that our formulations will lead to the study of a whole class of previously untapped therapeutics that may generate new solutions for previously untreatable diseases.

  13. Aberrant Chloride Intracellular Channel 4 Expression Contributes to Endothelial Dysfunction in Pulmonary Arterial Hypertension

    Science.gov (United States)

    Wojciak-Stothard, Beata; Abdul-Salam, Vahitha B.; Lao, Ka Hou; Tsang, Hilda; Irwin, David C.; Lisk, Christina; Loomis, Zoe; Stenmark, Kurt R.; Edwards, John C; Yuspa, Stuart H.; Howard, Luke S.; Edwards, Robert J.; Rhodes, Christopher J.; Gibbs, J Simon R.; Wharton, John; Zhao, Lan; Wilkins, Martin R.

    2014-01-01

    Background Chloride intracellular channel 4 (CLIC4) is highly expressed in the endothelium of remodelled pulmonary vessels and plexiform lesions of patients with pulmonary arterial hypertension (PAH). CLIC4 regulates vasculogenesis through endothelial tube formation. Aberrant CLIC4 expression may contribute to the vascular pathology of PAH. Methods and Results CLIC4 protein expression was increased in plasma and blood-derived endothelial cells from patients with idiopathic PAH (IPAH) and in the pulmonary vascular endothelium of 3 rat models of pulmonary hypertension. CLIC4 gene deletion markedly attenuated the development of chronic hypoxia-induced pulmonary hypertension in mice. Adenoviral overexpression of CLIC4 in cultured human pulmonary artery endothelial cells compromised pulmonary endothelial barrier function and enhanced their survival and angiogenic capacity, while CLIC4 shRNA had an inhibitory effect. Similarly, inhibition of CLIC4 expression in blood-derived endothelial cells from patients with IPAH attenuated the abnormal angiogenic behaviour that characterises these cells. The mechanism of CLIC4 effects involves p65-mediated activation of nuclear factor-κB, followed by stabilisation of hypoxia-inducible factor-1α and increased downstream production of vascular endothelial growth factor and endothelin-1. Conclusions Increased CLIC4 expression is an early manifestation and mediator of endothelial dysfunction in pulmonary hypertension. PMID:24503951

  14. Two-dimensional polyacrylamide gel electrophoresis of intracellular proteins

    International Nuclear Information System (INIS)

    Ojima, N.; Sakamoto, T.; Yamashita, M.

    1996-01-01

    Since two-dimensional electrophoresis was established by O'Farrell for analysis of intracellular proteins of Escherichia coli, it has been applied to separation of proteins of animal cells and tissues, and especially to identification of stress proteins. Using this technique, proteins are separated by isoelectric focusing containing 8 m urea in the first dimension and by SDS-PAGE in the second dimension. The gels are stained with Coomassie Blue R-250 dye, followed by silver staining. In the case of radio-labeled proteins, the gels are dried and then autoradiographed. In order to identify a specific protein separated by two-dimensional electrophoresis, a technique determining the N-terminal amino acid sequence of the protein has been developed recently. After the proteins in the gel were electrotransferred to a polyvinylidene difluoride membrane, the membrane was stained for protein with Commassie Blue and a stained membrane fragment was applied to a protein sequencer. Our recent studies demonstrated that fish cells newly synthesized various proteins in response to heat shock, cold nd osmotic stresses. For example, when cellular proteins extracted from cold-treated rainbow trout cells were subjected to two-dimensional gel electrophoresis, the 70 kDa protein was found to be synthesized during the cold-treatment. N-Terminal sequence analysis showed that the cold-inducible protein was a homolog of mammalian valosin-containing protein and yeast cell division cycle gene product CDC48p. Furthermore, the sequence data were useful for preparing PCR primers and a rabbit antibody against a synthetic peptide to analyze a role for the protein in the function of trout cells and mechanisms for regulation

  15. Eps15: a multifunctional adaptor protein regulating intracellular trafficking

    Directory of Open Access Journals (Sweden)

    van Bergen en Henegouwen Paul MP

    2009-10-01

    Full Text Available Abstract Over expression of receptor tyrosine kinases is responsible for the development of a wide variety of malignancies. Termination of growth factor signaling is primarily determined by the down regulation of active growth factor/receptor complexes. In recent years, considerable insight has been gained in the endocytosis and degradation of growth factor receptors. A crucial player in this process is the EGFR Protein tyrosine kinase Substrate #15, or Eps15. This protein functions as a scaffolding adaptor protein and is involved both in secretion and endocytosis. Eps15 has been shown to bind to AP-1 and AP-2 complexes, to bind to inositol lipids and to several other proteins involved in the regulation of intracellular trafficking. In addition, Eps15 has been detected in the nucleus of mammalian cells. Activation of growth factor receptors induces tyrosine phosphorylation and mono-ubiquitination of Eps15. The role of these post translational modifications of Eps15 is still a mystery. It is proposed that Eps15 and its family members Eps15R and Eps15b are involved in the regulation of membrane morphology, which is required for intracellular vesicle formation and trafficking.

  16. ATPase and GTPase Tangos Drive Intracellular Protein Transport.

    Science.gov (United States)

    Shan, Shu-Ou

    2016-12-01

    The GTPase superfamily of proteins provides molecular switches to regulate numerous cellular processes. The 'GTPase switch' paradigm, in which external regulatory factors control the switch of a GTPase between 'on' and 'off' states, has been used to interpret the regulatory mechanism of many GTPases. However, recent work unveiled a class of nucleotide hydrolases that do not adhere to this classical paradigm. Instead, they use nucleotide-dependent dimerization cycles to regulate key cellular processes. In this review article, recent studies of dimeric GTPases and ATPases involved in intracellular protein targeting are summarized. It is suggested that these proteins can use the conformational plasticity at their dimer interface to generate multiple points of regulation, thereby providing the driving force and spatiotemporal coordination of complex cellular pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Liposome-based Formulation for Intracellular Delivery of Functional Proteins

    Directory of Open Access Journals (Sweden)

    Benoît Chatin

    2015-01-01

    Full Text Available The intracellular delivery of biologically active protein represents an important emerging strategy for both fundamental and therapeutic applications. Here, we optimized in vitro delivery of two functional proteins, the β-galactosidase (β-gal enzyme and the anti-cytokeratin8 (K8 antibody, using liposome-based formulation. The guanidinium-cholesterol cationic lipid bis (guanidinium-tren-cholesterol (BGTC (bis (guanidinium-tren-cholesterol combined to the colipid dioleoyl phosphatidylethanolamine (DOPE (dioleoyl phosphatidylethanolamine was shown to efficiently deliver the β-gal intracellularly without compromising its activity. The lipid/protein molar ratio, protein amount, and culture medium were demonstrated to be key parameters affecting delivery efficiency. The protein itself is an essential factor requiring selection of the appropriate cationic lipid as illustrated by low K8 binding activity of the anti-K8 antibody using guanidinium-based liposome. Optimization of various lipids led to the identification of the aminoglycoside lipid dioleyl succinyl paromomycin (DOSP associated with the imidazole-based helper lipid MM27 as a potent delivery system for K8 antibody, achieving delivery in 67% of HeLa cells. Cryo-transmission electron microscopy showed that the structure of supramolecular assemblies BGTC:DOPE/β-gal and DOSP:MM27/K8 were different depending on liposome types and lipid/protein molar ratio. Finally, we observed that K8 treatment with DOSP:MM27/K8 rescues the cyclic adenosine monophosphate (cAMP-dependent chloride efflux in F508del-CFTR expressing cells, providing a new tool for the study of channelopathies.

  18. Dynamic intracellular localization of Dazl protein during Xenopus germline development.

    Science.gov (United States)

    Tada, Haru; Orii, Hidefumi

    2015-08-01

    Xenopus dazl encoding an RNA-binding protein has been identified as a component of the germ plasm and is involved in the migration and differentiation of the primordial germ cells (PGCs). Here, we investigated the intracellular localization of Dazl in germline cells throughout the lifetime of Xenopus. In early embryogenesis, Dazl was detected initially in the germ plasm and then translocated to a perinuclear region. Then, it was detected within the nucleus in PGCs. Dazl was observed only in the cytoplasm in PGCs when sex differentiation began in the gonads. Dazl was distributed in both the nucleus and cytoplasm of the primary oogonium and spermatogonium, but only in the cytoplasm of the secondary oogonium and spermatogonium. In spermatocytes, Dazl was distributed throughout cytoplasm and localized at the spindles and cytoplasm during meiosis. Then, it was detected as speckles in the nucleus in the round spermatid. The dynamic intracellular localization suggests that Dazl is a multifunctional protein regulating RNA metabolism required for Xenopus germline development.

  19. Intracellular distribution of cowpea mosaic virus movement protein as visualised by green fluorescent protein fusions

    NARCIS (Netherlands)

    Gopinath, K.; Bertens, P.; Pouwels, J.; Marks, H.; Lent, van J.W.M.; Wellink, J.E.; Kammen, van A.

    2003-01-01

    Cowpea mosaic virus (CPMV) derivatives expressing movement protein (MP) green fluorescent protein (GFP) fusions (MP:GFP) were used to study the intracellular targeting and localization of the MP in cowpea protoplasts and plants. In protoplasts, a virus coding for a wild type MP:GFP (MPfGFP) induced

  20. Intracellular Localization, Interactions and Functions of Capsicum Chlorosis Virus Proteins.

    Science.gov (United States)

    Widana Gamage, Shirani M K; Dietzgen, Ralf G

    2017-01-01

    Tospoviruses are among the most devastating viruses of horticultural and field crops. Capsicum chlorosis virus (CaCV) has emerged as an important pathogen of capsicum and tomato in Australia and South-east Asia. Present knowledge about CaCV protein functions in host cells is lacking. We determined intracellular localization and interactions of CaCV proteins by live plant cell imaging to gain insight into the associations of viral proteins during infection. Proteins were transiently expressed as fusions to autofluorescent proteins in leaf epidermal cells of Nicotiana benthamiana and capsicum. All viral proteins localized at least partially in the cell periphery suggestive of cytoplasmic replication and assembly of CaCV. Nucleocapsid (N) and non-structural movement (NSm) proteins localized exclusively in the cell periphery, while non-structural suppressor of silencing (NSs) protein and Gc and Gn glycoproteins accumulated in both the cell periphery and the nucleus. Nuclear localization of CaCV Gn and NSs is unique among tospoviruses. We validated nuclear localization of NSs by immunofluorescence in protoplasts. Bimolecular fluorescence complementation showed self-interactions of CaCV N, NSs and NSm, and heterotypic interactions of N with NSs and Gn. All interactions occurred in the cytoplasm, except NSs self-interaction was exclusively nuclear. Interactions of a tospoviral NSs protein with itself and with N had not been reported previously. Functionally, CaCV NSs showed strong local and systemic RNA silencing suppressor activity and appears to delay short-distance spread of silencing signal. Cell-to-cell movement activity of NSm was demonstrated by trans -complementation of a movement-defective tobamovirus replicon. CaCV NSm localized at plasmodesmata and its transient expression led to the formation of tubular structures that protruded from protoplasts. The D 155 residue in the 30K-like movement protein-specific LxD/N 50-70 G motif of NSm was critical for

  1. Wave failure at strong coupling in intracellular C a2 + signaling system with clustered channels

    Science.gov (United States)

    Li, Xiang; Wu, Yuning; Gao, Xuejuan; Cai, Meichun; Shuai, Jianwei

    2018-01-01

    As an important intracellular signal, C a2 + ions control diverse cellular functions. In this paper, we discuss the C a2 + signaling with a two-dimensional model in which the inositol 1,4,5-trisphosphate (I P3 ) receptor channels are distributed in clusters on the endoplasmic reticulum membrane. The wave failure at large C a2 + diffusion coupling is discussed in detail in the model. We show that with varying model parameters the wave failure is a robust behavior with either deterministic or stochastic channel dynamics. We suggest that the wave failure should be a general behavior in inhomogeneous diffusing systems with clustered excitable regions and may occur in biological C a2 + signaling systems.

  2. Intracellular Ca2+ and the phospholipid PIP2 regulate the taste transduction ion channel TRPM5

    OpenAIRE

    Liu, Dan; Liman, Emily R.

    2003-01-01

    The transduction of taste is a fundamental process that allows animals to discriminate nutritious from noxious substances. Three taste modalities, bitter, sweet, and amino acid, are mediated by G protein-coupled receptors that signal through a common transduction cascade: activation of phospholipase C β2, leading to a breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol and inositol 1,4,5-trisphosphate, which causes release of Ca2+ from intracellular stores. The ion c...

  3. Distribution of voltage-dependent and intracellular Ca2+ channels in submucosal neurons from rat distal colon.

    Science.gov (United States)

    Rehn, Matthias; Bader, Sandra; Bell, Anna; Diener, Martin

    2013-09-01

    We recently observed a bradykinin-induced increase in the cytosolic Ca2+ concentration in submucosal neurons of rat colon, an increase inhibited by blockers of voltage-dependent Ca2+ (Ca(v)) channels. As the types of Ca(v) channels used by this part of the enteric nervous system are unknown, the expression of various Ca(v) subunits has been investigated in whole-mount submucosal preparations by immunohistochemistry. Submucosal neurons, identified by a neuronal marker (microtubule-associated protein 2), are immunoreactive for Ca(v)1.2, Ca(v)1.3 and Ca(v)2.2, expression being confirmed by reverse transcription plus the polymerase chain reaction. These data agree with previous observations that the inhibition of L- and N-type Ca2+ currents strongly inhibits the response to bradykinin. However, whole-cell patch-clamp experiments have revealed that bradykinin does not enhance Ca2+ inward currents under voltage-clamp conditions. Consequently, bradykinin does not directly interact with Ca(v) channels. Instead, the kinin-induced Ca2+ influx is caused indirectly by the membrane depolarization evoked by this peptide. As intracellular Ca2+ channels on Ca(2+)-storing organelles can also contribute to Ca2+ signaling, their expression has been investigated by imaging experiments and immunohistochemistry. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) have been functionally demonstrated in submucosal neurons loaded with the Ca(2+)-sensitive fluorescent dye, fura-2. Histamine, a typical agonist coupled to the phospholipase C pathway, induces an increase in the fura-2 signal ratio, which is suppressed by 2-aminophenylborate, a blocker of IP3 receptors. The expression of IP3R1 has been confirmed by immunohistochemistry. In contrast, ryanodine, tested over a wide concentration range, evokes no increase in the cytosolic Ca2+ concentration nor is there immunohistochemical evidence for the expression of ryanodine receptors in these neurons. Thus, rat submucosal neurons are equipped

  4. Functionalized linear poly(amidoamine)s are efficient vectors for intracellular protein delivery

    NARCIS (Netherlands)

    Coué, G.M.J.P.C.; Engbersen, Johannes F.J.

    2011-01-01

    An effective intracellular protein delivery system was developed based on functionalized linear poly(amidoamine)s (PAAs) that form self-assembled cationic nanocomplexes with oppositely charged proteins. Three differently functionalized PAAs were synthesized, two of these having repetitive disulfide

  5. Intracellular Calcium Mobilization in Response to Ion Channel Regulators via a Calcium-Induced Calcium Release Mechanism

    OpenAIRE

    Petrou, Terry; Olsen, Herv?r L.; Thrasivoulou, Christopher; Masters, John R.; Ashmore, Jonathan F.; Ahmed, Aamir

    2017-01-01

    Free intracellular calcium ([Ca2+]i), in addition to being an important second messenger, is a key regulator of many cellular processes including the cell membrane potential, proliferation and apoptosis. In many cases, the mobilization of [Ca2+]i is controlled by intracellular store activation and calcium influx. We have investigated the effect of several ion channel modulators, which have been used to treat a range of human diseases, on [Ca2+]i release, by ratiometric calcium imaging. We sho...

  6. Trafficking and intracellular regulation of Kv7.1 potassium channels in the heart

    DEFF Research Database (Denmark)

    Nielsen, Nathalie Hélix

    The electrical activity of the heart, measured by application of surface body electrodes and recorded as an electrocardiogram, is the result of a finely tuned balance of ion movement (K+, Na+, Ca2+). The ionic currents collectively constitute the cardiac action potential created in the cell....... The mutation is located in the C-terminus of the Kv7.1 channel protein in a calmodulin binding domain, where the methionine (M) at position 520 is replaced by an arginine (R). Our results show that although Kv7.1/calmodulin interaction is not impaired by the M520R mutation, the mutated channels are retained...

  7. Structural Reconstruction of Protein-Protein Complexes Involved in Intracellular Signaling.

    Science.gov (United States)

    Kirsch, Klára; Sok, Péter; Reményi, Attila

    2016-01-01

    Signaling complexes within the cell convert extracellular cues into physiological outcomes. Their assembly involves signaling enzymes, allosteric regulators and scaffold proteins that often contain long stretches of disordered protein regions, display multi-domain architectures, and binding affinity between individual components is low. These features are indispensable for their central roles as dynamic information processing hubs, on the other hand they also make reconstruction of structurally homogeneous complex samples highly challenging. In this present chapter we discuss protein machinery which influences extracellular signal reception, intracellular pathway activity, and cytoskeletal or transcriptional activity.

  8. Silica Nanoparticles for Intracellular Protein Delivery: a Novel Synthesis Approach Using Green Fluorescent Protein

    Science.gov (United States)

    Schmidt, Sarah; Tavernaro, Isabella; Cavelius, Christian; Weber, Eva; Kümper, Alexander; Schmitz, Carmen; Fleddermann, Jana; Kraegeloh, Annette

    2017-09-01

    In this study, a novel approach for preparation of green fluorescent protein (GFP)-doped silica nanoparticles with a narrow size distribution is presented. GFP was chosen as a model protein due to its autofluorescence. Protein-doped nanoparticles have a high application potential in the field of intracellular protein delivery. In addition, fluorescently labelled particles can be used for bioimaging. The size of these protein-doped nanoparticles was adjusted from 15 to 35 nm using a multistep synthesis process, comprising the particle core synthesis followed by shell regrowth steps. GFP was selectively incorporated into the silica matrix of either the core or the shell or both by a one-pot reaction. The obtained nanoparticles were characterised by determination of particle size, hydrodynamic diameter, ζ-potential, fluorescence and quantum yield. The measurements showed that the fluorescence of GFP was maintained during particle synthesis. Cellular uptake experiments demonstrated that the GFP-doped nanoparticles can be used as stable and effective fluorescent probes. The study reveals the potential of the chosen approach for incorporation of functional biological macromolecules into silica nanoparticles, which opens novel application fields like intracellular protein delivery.

  9. Proteomic Analysis of Intracellular and Membrane Proteins From Voriconazole-Resistant Candida glabrata

    OpenAIRE

    Yoo, Jae Il; Kim, Hwa Su; Choi, Chi Won; Yoo, Jung Sik; Yu, Jae Yon; Lee, Yeong Seon

    2013-01-01

    Objectives The proteomic analysis of voriconazole resistant Candida glabrata strain has not yet been investigated. In this study, differentially expressed proteins of intracellular and membrane fraction from voriconazole-susceptible, susceptible dose-dependent (S-DD), resistant C. glabrata strains were compared with each other and several proteins were identified. Methods The proteins of intracellular and membrane were isolated by disrupting cells with glass bead and centrifugation from voric...

  10. Detection of Intracellular Factor VIII Protein in Peripheral Blood Mononuclear Cells by Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Gouri Shankar Pandey

    2013-01-01

    Full Text Available Flow cytometry is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. Detection of specific host proteins for diagnosis predominantly uses quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based detection assay for Factor VIII protein in peripheral blood mononuclear cells (PBMCs. An indirect intracellular staining (ICS method was standardized using monoclonal antibodies to different domains of human Factor VIII protein. The FVIII protein expression level was estimated by calculating the mean and median fluorescence intensities (MFI values for each monoclonal antibody. ICS staining of transiently transfected cell lines supported the method's specificity. Intracellular FVIII protein expression was also detected by the monoclonal antibodies used in the study in PBMCs of five blood donors. In summary, our data suggest that intracellular FVIII detection in PBMCs of hemophilia A patients can be a rapid and reliable method to detect intracellular FVIII levels.

  11. [The intracellular localization of the regulatory proteins of the densovirus of German cockroach, Blattella germanica].

    Science.gov (United States)

    Martynova, E U; Kapelinskaia, T V; Schal, C; Mukha, D V

    2014-01-01

    The intracellular localization of the regulatory proteins encoded by the genome of the densovirus of German cockroach was analyzed using western-blotting of nuclear and cytoplasmic extracts of the densovirus-infected passaging cells tissue culture BGE-2. Two of the three regulatory proteins, NS1 and NS3, were shown to possess mainly nuclear localization, while NS2 protein was distributed between the nucleus and cytoplasm. Data obtained provide new information necessary for prediction of the functions of densovirus regulatory proteins. Intracellular localization of NS3 protein was described for the densoviruses for the first time.

  12. Genetic analysis of hyperemesis gravidarum reveals association with intracellular calcium release channel (RYR2).

    Science.gov (United States)

    Fejzo, Marlena Schoenberg; Myhre, Ronny; Colodro-Conde, Lucía; MacGibbon, Kimber W; Sinsheimer, Janet S; Reddy, M V Prasad Linga; Pajukanta, Päivi; Nyholt, Dale R; Wright, Margaret J; Martin, Nicholas G; Engel, Stephanie M; Medland, Sarah E; Magnus, Per; Mullin, Patrick M

    2017-01-05

    Hyperemesis Gravidarum (HG), severe nausea/vomiting in pregnancy (NVP), can cause poor maternal/fetal outcomes. Genetic predisposition suggests the genetic component is essential in discovering an etiology. We performed whole-exome sequencing of 5 families followed by analysis of variants in 584 cases/431 controls. Variants in RYR2 segregated with disease in 2 families. The novel variant L3277R was not found in any case/control. The rare variant, G1886S was more common in cases (p = 0.046) and extreme cases (p = 0.023). Replication of G1886S using Norwegian/Australian data was supportive. Common variants rs790899 and rs1891246 were significantly associated with HG and weight loss. Copy-number analysis revealed a deletion in a patient. RYR2 encodes an intracellular calcium release channel involved in vomiting, cyclic-vomiting syndrome, and is a thyroid hormone target gene. Additionally, RYR2 is a downstream drug target of Inderal, used to treat HG and CVS. Thus, herein we provide genetic evidence for a pathway and therapy for HG. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. Intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly

    NARCIS (Netherlands)

    Horzinek, M.C.; Vennema, H.; Heijnen, L.; Zijderveld, A.; Spaan, W.J.M.

    1990-01-01

    Coronavirus spike protein genes were expressed in vitro by using the recombinant vaccinia virus expression system. Recombinant spike proteins were expressed at the cell surface and induced cell fusion in a host-cell-dependent fashion. The intracellular transport of recombinant spike proteins was

  14. How cholesterol interacts with proteins and lipids during its intracellular transport

    DEFF Research Database (Denmark)

    Wüstner, Daniel; Solanko, Katarzyna

    2015-01-01

    Sterols, as cholesterol in mammalian cells and ergosterol in fungi, are indispensable molecules for proper functioning and nanoscale organization of the plasma membrane. Synthesis, uptake and efflux of cholesterol are regulated by a variety of protein-lipid and protein-protein interactions. Simil...... specific protein-lipid and protein-protein interactions help overcoming the extremely low water solubility of cholesterol, thereby controlling intracellular cholesterol movement. This article is part of a Special Issue entitled: Lipid-protein interactions.......Sterols, as cholesterol in mammalian cells and ergosterol in fungi, are indispensable molecules for proper functioning and nanoscale organization of the plasma membrane. Synthesis, uptake and efflux of cholesterol are regulated by a variety of protein-lipid and protein-protein interactions....... Similarly, membrane lipids and their physico-chemical properties directly affect cholesterol partitioning and thereby contribute to the highly heterogeneous intracellular cholesterol distribution. Movement of cholesterol in cells is mediated by vesicle trafficking along the endocytic and secretory pathways...

  15. Proteomic Analysis of Intracellular and Membrane Proteins From Voriconazole-Resistant Candida glabrata.

    Science.gov (United States)

    Yoo, Jae Il; Kim, Hwa Su; Choi, Chi Won; Yoo, Jung Sik; Yu, Jae Yon; Lee, Yeong Seon

    2013-12-01

    The proteomic analysis of voriconazole resistant Candida glabrata strain has not yet been investigated. In this study, differentially expressed proteins of intracellular and membrane fraction from voriconazole-susceptible, susceptible dose-dependent (S-DD), resistant C. glabrata strains were compared with each other and several proteins were identified. The proteins of intracellular and membrane were isolated by disrupting cells with glass bead and centrifugation from voriconazole susceptible, S-DD, and resistant C. glabrata strains. The abundance of expressed proteins was compared using two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and proteins showing continuous twofold or more increase or reduction of expression in resistant strains compared to susceptible and S-DD strain were analyzed by liquid chromatography/mass spectrometry-mass spectrometry method. Of 34 intracellular proteins, 15 proteins showed expression increase or reduction (twofold or more). The identified proteins included regulation, energy production, carbohydrate transport, amino acid transport, and various metabolism related proteins. The increase of expression of heat shock protein 70 was found. Among membrane proteins, 12, 31 proteins showed expression increase or decrease in the order of susceptible, S-DD, and resistant strains. This expression included carbohydrate metabolism, amino acid synthesis, and response to stress-related proteins. In membrane fractions, the change of expression of 10 heat shock proteins was observed, and 9 heat shock protein 70 (Hsp70) showed the reduction of expression. The expression of Hsp70 protein in membrane fraction is related to voriconazole resistant C. glabrata strains.

  16. Intracellular calcium oscillations in strongly metastatic human breast and prostate cancer cells: control by voltage-gated sodium channel activity.

    Science.gov (United States)

    Rizaner, Nahit; Onkal, Rustem; Fraser, Scott P; Pristerá, Alessandro; Okuse, Kenji; Djamgoz, Mustafa B A

    2016-10-01

    The possible association of intracellular Ca 2+ with metastasis in human cancer cells is poorly understood. We have studied Ca 2+ signaling in human prostate and breast cancer cell lines of strongly versus weakly metastatic potential in a comparative approach. Intracellular free Ca 2+ was measured using a membrane-permeant fluorescent Ca 2+ -indicator dye (Fluo-4 AM) and confocal microscopy. Spontaneous Ca 2+ oscillations were observed in a proportion of strongly metastatic human prostate and breast cancer cells (PC-3M and MDA-MB-231, respectively). In contrast, no such oscillations were observed in weakly/non metastatic LNCaP and MCF-7 cells, although a rise in the resting Ca 2+ level could be induced by applying a high-K + solution. Various parameters of the oscillations depended on extracellular Ca 2+ and voltage-gated Na + channel activity. Treatment with either tetrodotoxin (a general blocker of voltage-gated Na + channels) or ranolazine (a blocker of the persistent component of the channel current) suppressed the Ca 2+ oscillations. It is concluded that the functional voltage-gated Na + channel expression in strongly metastatic cancer cells makes a significant contribution to generation of oscillatory intracellular Ca 2+ activity. Possible mechanisms and consequences of the Ca 2+ oscillations are discussed.

  17. Protective functions of intracellular heat-shock protein (HSP) 70-expression in patients with severe sepsis.

    Science.gov (United States)

    Bruemmer-Smith, S; Stüber, F; Schroeder, S

    2001-12-01

    Intracellular expression of heat-shock-protein 70 (HSP70) arose early in evolutionary development as a tool to protect cellular homeostasis. HSP70 detects proteins that are incorrectly folded or denatured. They form a complex with such proteins which can lead to correct folding, compartmentalization in organelles, or to proteolytic degradation. HSP70 also appears to protect proteins from degeneration. Intracellular HSP70-expression is induced by a wide variety of stimuli including heat, fever, hypoxia, oxygen radicals, endotoxins, cytokines, and heavy metal ions. Pre-emptive induction of HSP70-expression reduces organ dysfunction and mortality in animal models of sepsis.

  18. Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Overton, K. Wesley; Park, Dan M.; Yung, Mimi C.; Dohnalkova, Alice; Smit, John; Jiao, Yongqin

    2016-09-23

    ABSTRACT

    Surface layers, or S-layers, are two-dimensional protein arrays that form the outermost layer of many bacteria and archaea. They serve several functions, including physical protection of the cell from environmental threats. The high abundance of S-layer proteins necessitates a highly efficient export mechanism to transport the S-layer protein from the cytoplasm to the cell exterior.Caulobacter crescentusis unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaFaand RsaFb, which together with other components form a type I protein translocation pathway for S-layer export. These proteins have homology toEscherichia coliTolC, the outer membrane channel of multidrug efflux pumps. Here we provide evidence that, unlike TolC, RsaFaand RsaFbare not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaFaand RsaFbare required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaFaand RsaFbled to a general growth defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and transcriptome sequencing (RNA-seq), we show that loss of both RsaFaand RsaFbled to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involved in protein misfolding and degradation pathways. These findings provide new insight into the requirement for RsaFaand RsaFbin cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels in

  19. Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production.

    Directory of Open Access Journals (Sweden)

    Ann L Wozniak

    2010-09-01

    Full Text Available The hepatitis C virus (HCV p7 protein is critical for virus production and an attractive antiviral target. p7 is an ion channel when reconstituted in artificial lipid bilayers, but channel function has not been demonstrated in vivo and it is unknown whether p7 channel activity plays a critical role in virus production. To evaluate the contribution of p7 to organelle pH regulation and virus production, we incorporated a fluorescent pH sensor within native, intracellular vesicles in the presence or absence of p7 expression. p7 increased proton (H(+ conductance in vesicles and was able to rapidly equilibrate H(+ gradients. This conductance was blocked by the viroporin inhibitors amantadine, rimantadine and hexamethylene amiloride. Fluorescence microscopy using pH indicators in live cells showed that both HCV infection and expression of p7 from replicon RNAs reduced the number of highly acidic (pH<5 vesicles and increased lysosomal pH from 4.5 to 6.0. These effects were not present in uninfected cells, sub-genomic replicon cells not expressing p7, or cells electroporated with viral RNA containing a channel-inactive p7 point mutation. The acidification inhibitor, bafilomycin A1, partially restored virus production to cells electroporated with viral RNA containing the channel inactive mutation, yet did not in cells containing p7-deleted RNA. Expression of influenza M2 protein also complemented the p7 mutant, confirming a requirement for H(+ channel activity in virus production. Accordingly, exposure to acid pH rendered intracellular HCV particles non-infectious, whereas the infectivity of extracellular virions was acid stable and unaffected by incubation at low pH, further demonstrating a key requirement for p7-induced loss of acidification. We conclude that p7 functions as a H(+ permeation pathway, acting to prevent acidification in otherwise acidic intracellular compartments. This loss of acidification is required for productive HCV infection

  20. Intracellular Delivery of Proteins via Fusion Peptides in Intact Plants.

    Directory of Open Access Journals (Sweden)

    Kiaw Kiaw Ng

    Full Text Available In current plant biotechnology, the introduction of exogenous DNA encoding desired traits is the most common approach used to modify plants. However, general plant transformation methods can cause random integration of exogenous DNA into the plant genome. To avoid these events, alternative methods, such as a direct protein delivery system, are needed to modify the plant. Although there have been reports of the delivery of proteins into cultured plant cells, there are currently no methods for the direct delivery of proteins into intact plants, owing to their hierarchical structures. Here, we demonstrate the efficient fusion-peptide-based delivery of proteins into intact Arabidopsis thaliana. Bovine serum albumin (BSA, 66 kDa was selected as a model protein to optimize conditions for delivery into the cytosol. The general applicability of our method to large protein cargo was also demonstrated by the delivery of alcohol dehydrogenase (ADH, 150 kDa into the cytosol. The compatibility of the fusion peptide system with the delivery of proteins to specific cellular organelles was also demonstrated using the fluorescent protein Citrine (27 kDa conjugated to either a nuclear localization signal (NLS or a peroxisomal targeting signal (PTS. In conclusion, our designed fusion peptide system can deliver proteins with a wide range of molecular weights (27 to 150 kDa into the cells of intact A. thaliana without interfering with the organelle-targeting peptide conjugated to the protein. We expect that this efficient protein delivery system will be a powerful tool in plant biotechnology.

  1. Cytoplasmic tail of coronavirus spike protein has intracellular ...

    Indian Academy of Sciences (India)

    2017-04-18

    Apr 18, 2017 ... protein, hemagglutinin from the influenza virus, sorts to the apical domain (Mora et al. 2002). To mediate budding ..... After 48 h of post-transfection, cell was immunolabelled using rabbit anti-HCoV-SARS S protein antisera for SARS-S-Y construct (b) and rabbit anti-BCV antisera for OC43-S-Y construct (e) ...

  2. Bioreducible poly(amidoamine)s as carriers for intracellular protein delivery to intestinal cells

    NARCIS (Netherlands)

    Cohen, S.; Coué, G.M.J.P.C.; Beno, D.; Korenstein, R.; Engbersen, Johannes F.J.

    2012-01-01

    An effective intracellular protein delivery system was developed based on linear poly(amidoamine)s (PAAs) that form self-assembled cationic nanocomplexes with oppositely charged proteins. Two differently functionalized PAAs were synthesized by Michael-type polyaddition of 4-amino-1-butanol (ABOL) to

  3. Intracellular localization of ornithine decarboxylase and its regulatory protein, antizyme-1.

    NARCIS (Netherlands)

    Schipper, R.G.; Cuijpers, V.M.J.I.; Groot, L.H. de; Thio, M.; Verhofstad, A.A.J.

    2004-01-01

    The enzyme ornithine decarboxylase (ODC) and its regulatory protein antizyme-1 (AZ1) are key regulators in the homeostasis of polyamines. To gain more insight into the exact intracellular distribution of ODC and AZ1, we performed immunocytochemical and Green Fluorescent Protein-fluorocytochemical

  4. The DNAJB6 and DNAJB8 protein chaperones prevent intracellular aggregation of polyglutamine peptides

    NARCIS (Netherlands)

    Gillis, J.; Schipper-Krom, S.; Juenemann, K.; Gruber, A.; Coolen, S.; van den Nieuwendijk, R.; van Veen, H.; Overkleeft, H.; Goedhart, J.; Kampinga, H.H.; Reits, E.A.

    2013-01-01

    Fragments of proteins containing an expanded polyglutamine (polyQ) tract are thought to initiate aggregation and toxicity in at least nine neurodegenerative diseases, including Huntington's disease. Because proteasomes appear unable to digest the polyQ tract, which can initiate intracellular protein

  5. The DNAJB6 and DNAJB8 Protein Chaperones Prevent Intracellular Aggregation of Polyglutamine Peptides

    NARCIS (Netherlands)

    Gillis, Judith; Schipper-Krom, Sabine; Juenemann, Katrin; Gruber, Anna; Coolen, Silvia; van den Nieuwendijk, Rian; van Veen, Henk; Overkleeft, Hermen; Goedhart, Joachim; Kampinga, Harm H.; Reits, Eric A.

    2013-01-01

    Fragments of proteins containing an expanded polyglutamine (polyQ) tract are thought to initiate aggregation and toxicity in at least nine neurodegenerative diseases, including Huntington's disease. Because proteasomes appear unable to digest the polyQ tract, which can initiate intracellular protein

  6. Controlled release and intracellular protein delivery from mesoporous silica nanoparticles.

    Science.gov (United States)

    Deodhar, Gauri V; Adams, Marisa L; Trewyn, Brian G

    2017-01-01

    Protein therapeutics are promising candidates for disease treatment due to their high specificity and minimal adverse side effects; however, targeted protein delivery to specific sites has proven challenging. Mesoporous silica nanoparticles (MSN) have demonstrated to be ideal candidates for this application, given their high loading capacity, biocompatibility, and ability to protect host molecules from degradation. These materials exhibit tunable pore sizes, shapes and volumes, and surfaces which can be easily functionalized. This serves to control the movement of molecules in and out of the pores, thus entrapping guest molecules until a specific stimulus triggers release. In this review, we will cover the benefits of using MSN as protein therapeutic carriers, demonstrating that there is great diversity in the ways MSN can be used to service proteins. Methods for controlling the physical dimensions of pores via synthetic conditions, applications of therapeutic protein loaded MSN materials in cancer therapies, delivering protein loaded MSN materials to plant cells using biolistic methods, and common stimuli-responsive functionalities will be discussed. New and exciting strategies for controlled release and manipulation of proteins are also covered in this review. While research in this area has advanced substantially, we conclude this review with future challenges to be tackled by the scientific community. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Rab proteins: The key regulators of intracellular vesicle transport

    International Nuclear Information System (INIS)

    Bhuin, Tanmay; Roy, Jagat Kumar

    2014-01-01

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future

  8. Rab proteins: The key regulators of intracellular vesicle transport

    Energy Technology Data Exchange (ETDEWEB)

    Bhuin, Tanmay [Cell and Developmental Biology Unit, Department of Zoology, The University of Burdwan, Golapbag 713104 (India); Roy, Jagat Kumar, E-mail: jkroy@bhu.ac.in [Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005 (India)

    2014-10-15

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.

  9. Endocytosis and intracellular protein degradation in cystic fibrosis fibroblasts

    International Nuclear Information System (INIS)

    Jessup, W.; Dean, R.T.

    1983-01-01

    Normal rates of pinocytosis of [ 3 H]sucrose were measured in cystic fibrosis fibroblasts, and were not affected by the addition of cystic fibrosis serum. Bulk protein degradation (a significant proportion of which occurs intralysosomally following autophagy) and its regulation by growth state were apparently identical in normal and cystic fibrosis cultures. (Auth.)

  10. Cytoplasmic tail of Coronavirus spike protein has intracellular ...

    Indian Academy of Sciences (India)

    58

    Transfection ability of YFP tagged spike protein constructs are much more efficient. 220 compared to wild type spike construct, the reasons for which are unclear (data not. 221 shown). Because of efficient detection of YFP fluorescence and the limitations of spike. 222 specific antibodies, we decided to use the YFP tagged ...

  11. Post-translational selective intracellular silencing of acetylated proteins with de novo selected intrabodies.

    Science.gov (United States)

    Chirichella, Michele; Lisi, Simonetta; Fantini, Marco; Goracci, Martina; Calvello, Mariantonietta; Brandi, Rossella; Arisi, Ivan; D'Onofrio, Mara; Di Primio, Cristina; Cattaneo, Antonino

    2017-03-01

    The ability to selectively interfere with post-translationally modified proteins would have many biological and therapeutic applications. However, post-translational modifications cannot be selectively targeted by nucleic-acid-based interference approaches. Here we describe post-translational intracellular silencing antibody technology (PISA), a method for selecting intrabodies against post-translationally modified proteins. We demonstrate our method by generating intrabodies against native acetylated proteins and showing functional interference in living cells.

  12. Minireview: Role of Intracellular Scaffolding Proteins in the Regulation of Endocrine G Protein-Coupled Receptor Signaling

    Science.gov (United States)

    Walther, Cornelia

    2015-01-01

    The majority of hormones stimulates and mediates their signal transduction via G protein-coupled receptors (GPCRs). The signal is transmitted into the cell due to the association of the GPCRs with heterotrimeric G proteins, which in turn activates an extensive array of signaling pathways to regulate cell physiology. However, GPCRs also function as scaffolds for the recruitment of a variety of cytoplasmic protein-interacting proteins that bind to both the intracellular face and protein interaction motifs encoded by GPCRs. The structural scaffolding of these proteins allows GPCRs to recruit large functional complexes that serve to modulate both G protein-dependent and -independent cellular signaling pathways and modulate GPCR intracellular trafficking. This review focuses on GPCR interacting PSD95-disc large-zona occludens domain containing scaffolds in the regulation of endocrine receptor signaling as well as their potential role as therapeutic targets for the treatment of endocrinopathies. PMID:25942107

  13. Altered intracellular localization and mobility of SBDS protein upon mutation in Shwachman-Diamond syndrome.

    Science.gov (United States)

    Orelio, Claudia; van der Sluis, Renée M; Verkuijlen, Paul; Nethe, Micha; Hordijk, Peter L; van den Berg, Timo K; Kuijpers, Taco W

    2011-01-01

    Shwachman-Diamond Syndrome (SDS) is a rare inherited disease caused by mutations in the SBDS gene. Hematopoietic defects, exocrine pancreas dysfunction and short stature are the most prominent clinical features. To gain understanding of the molecular properties of the ubiquitously expressed SBDS protein, we examined its intracellular localization and mobility by live cell imaging techniques. We observed that SBDS full-length protein was localized in both the nucleus and cytoplasm, whereas patient-related truncated SBDS protein isoforms localize predominantly to the nucleus. Also the nucleo-cytoplasmic trafficking of these patient-related SBDS proteins was disturbed. Further studies with a series of SBDS mutant proteins revealed that three distinct motifs determine the intracellular mobility of SBDS protein. A sumoylation motif in the C-terminal domain, that is lacking in patient SBDS proteins, was found to play a pivotal role in intracellular motility. Our structure-function analyses provide new insight into localization and motility of the SBDS protein, and show that patient-related mutant proteins are altered in their molecular properties, which may contribute to the clinical features observed in SDS patients.

  14. Altered intracellular localization and mobility of SBDS protein upon mutation in Shwachman-Diamond syndrome.

    Directory of Open Access Journals (Sweden)

    Claudia Orelio

    Full Text Available Shwachman-Diamond Syndrome (SDS is a rare inherited disease caused by mutations in the SBDS gene. Hematopoietic defects, exocrine pancreas dysfunction and short stature are the most prominent clinical features. To gain understanding of the molecular properties of the ubiquitously expressed SBDS protein, we examined its intracellular localization and mobility by live cell imaging techniques. We observed that SBDS full-length protein was localized in both the nucleus and cytoplasm, whereas patient-related truncated SBDS protein isoforms localize predominantly to the nucleus. Also the nucleo-cytoplasmic trafficking of these patient-related SBDS proteins was disturbed. Further studies with a series of SBDS mutant proteins revealed that three distinct motifs determine the intracellular mobility of SBDS protein. A sumoylation motif in the C-terminal domain, that is lacking in patient SBDS proteins, was found to play a pivotal role in intracellular motility. Our structure-function analyses provide new insight into localization and motility of the SBDS protein, and show that patient-related mutant proteins are altered in their molecular properties, which may contribute to the clinical features observed in SDS patients.

  15. TRP channel-associated factors are a novel protein family that regulates TRPM8 trafficking and activity.

    NARCIS (Netherlands)

    Gkika, D.; Lemonnier, L.; Shapovalov, G.; Gordienko, D.; Poux, C.; Bernardini, M.; Bokhobza, A.; Bidaux, G.; Degerny, C.; Verreman, K.; Guarmit, B.; Benahmed, M.; Launoit, Y. de; Bindels, R.J.M.; Fiorio Pla, A.; Prevarskaya, N.

    2015-01-01

    TRPM8 is a cold sensor that is highly expressed in the prostate as well as in other non-temperature-sensing organs, and is regulated by downstream receptor-activated signaling pathways. However, little is known about the intracellular proteins necessary for channel function. Here, we identify two

  16. Intracellular oxygen determined by respiration regulates localization of Ras and prenylated proteins.

    Science.gov (United States)

    Kim, A; Davis, R; Higuchi, M

    2015-07-16

    Reduction of mitochondrial DNA (mtDNA) content induces the reduction of oxidative phosphorylation and dependence on fermentative glycolysis, that is, the Warburg effect. In aggressive prostate cancer (PCa), the reduction of mtDNA reduces oxygen consumption, increases intracellular oxygen concentration, and induces constitutive activation of Ras. Many essential proteins for cell death, growth, differentiation, and development, such as Ras, require prenylation for subcellular localization and activation. Prenylation of a protein is defined as the attachment of isoprenoids to a cysteine residue at or near the C-terminus. 3-Hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) produces isoprenoids, and is posttranslationally regulated by oxygen. We investigated a critical role of intracellular oxygen in membrane localization of prenylated proteins. Localization of prenylated proteins (H-Ras, prelamin A/C, and Rab5a) was observed in poorly differentiated PCa (PC-3) and well-differentiated PCa (LNCaP) cells. PC-3 cells exhibited high intracellular oxygen concentration, and H-Ras, prelamin A/C, and Rab5a were localized to various membranes (Golgi and plasma membrane, nuclear membrane, and early endosomes, respectively). Remarkably, exogenous hypoxia (0.2% O2) in PC-3 cells induced intracellular hypoxia and changed the localization of the prenylated proteins. H-Ras and Rab5a were translocated to cytosol, and prelamin A/C was in the nucleus forming an abnormal nuclear envelope. The localization was reversed by mevalonate indicating the involvement of mevalonate pathway. In contrast, in LNCaP cells, exhibiting low intracellular oxygen concentration, H-Ras and Rab5a were localized in the cytosol, and prelamin A/C was inside the nucleus forming an inadequate nuclear envelope. Exogenous hyperoxia (40% O2) increased the intracellular oxygen concentration and induced Ras translocation from cytosol to the membrane. Prelamin A/C was translocated to the nuclear membrane and formed a

  17. Intracellular localisation of proteins to specific cellular areas by nanocapsule mediated delivery.

    Science.gov (United States)

    Wang, Huabin; Chen, Ligang; Sun, Xianchao; Fu, Ailing

    2017-09-01

    Nanocapsules are promising carriers with great potential for intracellular protein transport. Although many studies have intended to improve cell uptake efficacy, there is an increasing interest in understanding of subcellular distribution of cargoes inside cells, which is essential for purposeful delivery of biomolecules into specific sites within cells. Herein, we interrogate the intracellular localisation of exogenous proteins, including fluorescein isothiocyanate (FITC)-labelled bovine serum albumin (BSA) and green fluorescent protein (GFP), mediated by specially designed nanocapsules. The results show that the designed nanocapsules can deliver the two types of fluorescent proteins into different cellular destinations (cytosol, nucleus or the whole cell), depending on the composition of nanocapsules. Meanwhile, several impact factors that influence the distribution of proteins in cells have also been investigated, and the results suggest that the localisation of capsule-mediated proteins in cells is strongly affected by the surface properties of nanocapsules, the types of stabilisers and proteins, and environmental temperatures. The rational control of intracellular localised delivery of exogenous proteins as we demonstrated in this study might open new avenues to obtain desired magnitude of drug effects for modulating cell activity.

  18. Intra-membrane molecular interactions of K+ channel proteins :

    Energy Technology Data Exchange (ETDEWEB)

    Moczydlowski, Edward G.

    2013-07-01

    Ion channel proteins regulate complex patterns of cellular electrical activity and ionic signaling. Certain K+ channels play an important role in immunological biodefense mechanisms of adaptive and innate immunity. Most ion channel proteins are oligomeric complexes with the conductive pore located at the central subunit interface. The long-term activity of many K+ channel proteins is dependent on the concentration of extracellular K+; however, the mechanism is unclear. Thus, this project focused on mechanisms underlying structural stability of tetrameric K+ channels. Using KcsA of Streptomyces lividans as a model K+ channel of known structure, the molecular basis of tetramer stability was investigated by: 1. Bioinformatic analysis of the tetramer interface. 2. Effect of two local anesthetics (lidocaine, tetracaine) on tetramer stability. 3. Molecular simulation of drug docking to the ion conduction pore. The results provide new insights regarding the structural stability of K+ channels and its possible role in cell physiology.

  19. A selectivity filter at the intracellular end of the acid-sensing ion channel pore

    DEFF Research Database (Denmark)

    Lynagh, Timothy; Flood, Emelie; Boiteux, Céline

    2017-01-01

    Increased extracellular proton concentrations during neurotransmission are converted to excitatory sodium influx by acid-sensing ion channels (ASICs). 10-fold sodium/potassium selectivity in ASICs has long been attributed to a central constriction in the channel pore, but experimental verification...

  20. Increased Numbers of Ion Channels Promoted by an Intracellular Second Messenger

    Science.gov (United States)

    Gunning, Richard

    1987-01-01

    The anomalous rectifier potassium current in Aplysia neurons was examined to determine the immediate cause of an increase in conductance induced by serotonin and mediated by adenosine 3' ,5' -monophosphate. Voltage-dependent cesium ion block and steady-state current power spectral density were measured under voltage clamp before and after application of serotonin. The amplitude of the anomalous rectifier conductance was increased by adding serotonin, but the shapes of the conductance-voltage curve and the power spectrum were not altered. Calculation of the number of functional channels and of the single-channel conductance from the power spectra indicates that the serotonin-induced increase in conductance resulted from an increase in the number of functional channels, while the single-channel conductance and the open-channel probability were unchanged.

  1. Highly Efficient Intracellular Protein Delivery by Cationic Polyethyleneimine-Modified Gelatin Nanoparticles

    Directory of Open Access Journals (Sweden)

    Ming-Ju Chou

    2018-02-01

    Full Text Available Intracellular protein delivery may provide a safe and non-genome integrated strategy for targeting abnormal or specific cells for applications in cell reprogramming therapy. Thus, highly efficient intracellular functional protein delivery would be beneficial for protein drug discovery. In this study, we generated a cationic polyethyleneimine (PEI-modified gelatin nanoparticle and evaluated its intracellular protein delivery ability in vitro and in vivo. The experimental results showed that the PEI-modified gelatin nanoparticle had a zeta potential of approximately +60 mV and the particle size was approximately 135 nm. The particle was stable at different biological pH values and temperatures and high protein loading efficiency was observed. The fluorescent image results revealed that large numbers of particles were taken up into the mammalian cells and escaped from the endosomes into the cytoplasm. In a mouse C26 cell-xenograft cancer model, particles accumulated in cancer cells. In conclusion, the PEI-modified gelatin particle may provide a biodegradable and highly efficient protein delivery system for use in regenerative medicine and cancer therapy.

  2. Probing potassium channel function in vivo by intracellular delivery of antibodies in a rat model of retinal neurodegeneration

    Science.gov (United States)

    Raz-Prag, Dorit; Grimes, William N.; Fariss, Robert N.; Vijayasarathy, Camasamudram; Campos, Maria M.; Bush, Ronald A.; Diamond, Jeffrey S.; Sieving, Paul A.

    2010-01-01

    Inward rectifying potassium (Kir) channels participate in regulating potassium concentration (K+) in the central nervous system (CNS), including in the retina. We explored the contribution of Kir channels to retinal function by delivering Kir antibodies (Kir-Abs) into the rat eye in vivo to interrupt channel activity. Kir-Abs were coupled to a peptide carrier to reach intracellular epitopes. Functional effects were evaluated by recording the scotopic threshold response (STR) and photopic negative response (PhNR) of the electroretinogram (ERG) noninvasively with an electrode on the cornea to determine activity of the rod and cone pathways, respectively. Intravitreal delivery of Kir2.1-Ab coupled to the peptide carrier diminished these ERG responses equivalent to dimming the stimulus 10- to 100-fold. Immunohistochemistry (IHC) showed Kir2.1 immunostaining of retinal bipolar cells (BCs) matching the labeling pattern obtained with conventional IHC of applying Kir2.1-Ab to fixed retinal sections postmortem. Whole-cell voltage-clamp BC recordings in rat acute retinal slices showed suppression of barium-sensitive Kir2.1 currents upon inclusion of Kir2.1-Ab in the patch pipette. The in vivo functional and structural results implicate a contribution of Kir2.1 channel activity in these electronegative ERG potentials. Studies with Kir4.1-Ab administered in vivo also suppressed the ERG components and showed immunostaining of Müller cells. The strategy of administering Kir antibodies in vivo, coupled to a peptide carrier to facilitate intracellular delivery, identifies roles for Kir2.1 and Kir4.1 in ERG components arising in the proximal retina and suggests this approach could be of further value in research. PMID:20616020

  3. Cellular prion protein expression is not regulated by the Alzheimer's amyloid precursor protein intracellular domain.

    Directory of Open Access Journals (Sweden)

    Victoria Lewis

    Full Text Available There is increasing evidence of molecular and cellular links between Alzheimer's disease (AD and prion diseases. The cellular prion protein, PrP(C, modulates the post-translational processing of the AD amyloid precursor protein (APP, through its inhibition of the β-secretase BACE1, and oligomers of amyloid-β bind to PrP(C which may mediate amyloid-β neurotoxicity. In addition, the APP intracellular domain (AICD, which acts as a transcriptional regulator, has been reported to control the expression of PrP(C. Through the use of transgenic mice, cell culture models and manipulation of APP expression and processing, this study aimed to clarify the role of AICD in regulating PrP(C. Over-expression of the three major isoforms of human APP (APP(695, APP(751 and APP(770 in cultured neuronal and non-neuronal cells had no effect on the level of endogenous PrP(C. Furthermore, analysis of brain tissue from transgenic mice over-expressing either wild type or familial AD associated mutant human APP revealed unaltered PrP(C levels. Knockdown of endogenous APP expression in cells by siRNA or inhibition of γ-secretase activity also had no effect on PrP(C levels. Overall, we did not detect any significant difference in the expression of PrP(C in any of the cell or animal-based paradigms considered, indicating that the control of cellular PrP(C levels by AICD is not as straightforward as previously suggested.

  4. Ion channel regulation by protein S-acylation

    Science.gov (United States)

    2014-01-01

    Protein S-acylation, the reversible covalent fatty-acid modification of cysteine residues, has emerged as a dynamic posttranslational modification (PTM) that controls the diversity, life cycle, and physiological function of numerous ligand- and voltage-gated ion channels. S-acylation is enzymatically mediated by a diverse family of acyltransferases (zDHHCs) and is reversed by acylthioesterases. However, for most ion channels, the dynamics and subcellular localization at which S-acylation and deacylation cycles occur are not known. S-acylation can control the two fundamental determinants of ion channel function: (1) the number of channels resident in a membrane and (2) the activity of the channel at the membrane. It controls the former by regulating channel trafficking and the latter by controlling channel kinetics and modulation by other PTMs. Ion channel function may be modulated by S-acylation of both pore-forming and regulatory subunits as well as through control of adapter, signaling, and scaffolding proteins in ion channel complexes. Importantly, cross-talk of S-acylation with other PTMs of both cysteine residues by themselves and neighboring sites of phosphorylation is an emerging concept in the control of ion channel physiology. In this review, I discuss the fundamentals of protein S-acylation and the tools available to investigate ion channel S-acylation. The mechanisms and role of S-acylation in controlling diverse stages of the ion channel life cycle and its effect on ion channel function are highlighted. Finally, I discuss future goals and challenges for the field to understand both the mechanistic basis for S-acylation control of ion channels and the functional consequence and implications for understanding the physiological function of ion channel S-acylation in health and disease. PMID:24821965

  5. Asp3Gly polymorphism affects fatty acid-binding protein 3 intracellular stability and subcellular localization.

    Science.gov (United States)

    Kusudo, Tatsuya; Hashida, Yasuhiko; Ando, Fujiko; Shimokata, Hiroshi; Yamashita, Hitoshi

    2015-08-19

    Fatty acid-binding proteins (FABP) play a crucial role in intracellular fatty acid transportation and metabolism. In this study, we investigate the effects of the FABP3 Asp3Gly (D3G) polymorphism on protein structure and function. Although the mutation did not alter protein secondary structure or the ability to bind 1-anilinonaphthalene-8-sulfonic acid and palmitate, the intracellular stability of the D3G mutant was significantly decreased. Immunocytochemical analysis reveals that the mutation alters FABP3 subcellular localization. Our results suggest that the D3G polymorphism may impact energy metabolism and physiological functions. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  6. Intracellular Calcium Mobilization in Response to Ion Channel Regulators via a Calcium-Induced Calcium Release Mechanism.

    Science.gov (United States)

    Petrou, Terry; Olsen, Hervør L; Thrasivoulou, Christopher; Masters, John R; Ashmore, Jonathan F; Ahmed, Aamir

    2017-02-01

    Free intracellular calcium ([Ca 2+ ] i ), in addition to being an important second messenger, is a key regulator of many cellular processes including cell membrane potential, proliferation, and apoptosis. In many cases, the mobilization of [Ca 2+ ] i is controlled by intracellular store activation and calcium influx. We have investigated the effect of several ion channel modulators, which have been used to treat a range of human diseases, on [Ca 2+ ] i release, by ratiometric calcium imaging. We show that six such modulators [amiodarone (Ami), dofetilide, furosemide (Fur), minoxidil (Min), loxapine (Lox), and Nicorandil] initiate release of [Ca 2+ ] i in prostate and breast cancer cell lines, PC3 and MCF7, respectively. Whole-cell currents in PC3 cells were inhibited by the compounds tested in patch-clamp experiments in a concentration-dependent manner. In all cases [Ca 2+ ] i was increased by modulator concentrations comparable to those used clinically. The increase in [Ca 2+ ] i in response to Ami, Fur, Lox, and Min was reduced significantly (P calcium was reduced to nM concentration by chelation with EGTA. The data suggest that many ion channel regulators mobilize [Ca 2+ ] i We suggest a mechanism whereby calcium-induced calcium release is implicated; such a mechanism may be important for understanding the action of these compounds. Copyright © 2017 by The Author(s).

  7. Determination of Six Transmembrane Protein of Prostate 2 Gene Expression and Intracellular Localization in Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Bora İrer

    2017-12-01

    Full Text Available Objective: In this study, we aimed to determine the relationship between the RNA and protein expression profile of six transmembrane protein of prostate 2 (STAMP2 gene and androgen and the intracellular localization of STAMP2. Materials and Methods: RNA and protein were obtained from androgen treated lymph node carcinoma of the prostate (LNCaP cells, untreated LNCaP cells, DU145 cells with no androgen receptor, and STAMP2 transfected COS-7 cells. The expression profile of STAMP2 gene and the effect of androgenes on the expression was shown in RNA and protein levels by using Northern and Western blotting methods. In addition, intracellular localization of the naturally synthesized STAMP2 protein and the transfected STAMP2 protein in COS-7 cells after androgen administration in both LNCaP cells was determined by immunofluorescence microscopy. Results: We found that the RNA and protein expression of STAMP2 gene in LNCaP cells are regulated by androgenes, the power of expression is increased with the duration of androgen treatment and there is no STAMP2 expression in DU145 cells which has no androgen receptor. As a result of the immunofluorescence microscopy study we observed that STAMP2 protein was localized at golgi complex and cell membrane. Conclusion: In conclusion, we have demonstrated that STAMP2 may play an important role in the pathogenesis of the prostate cancer and in the androgen-dependent androgen-independent staging of prostate cancer. In addition, STAMP2 protein, which is localized in the intracellular golgi complex and cell membrane, may be a new target molecule for prostate cancer diagnosis and treatment.

  8. Effect of channel-protein interaction on translocation of a protein-like chain through a finite channel

    International Nuclear Information System (INIS)

    Sun Ting-Ting; Ma Hai-Zhu; Jiang Zhou-Ting

    2012-01-01

    We study the translocation of a protein-like chain through a finite cylindrical channel using the pruned-enriched Rosenbluth method (PERM) and the modified orientation-dependent monomer-monomer interaction (ODI) model. Attractive channels (in cp = −2.0, −1.0, −0.5), repulsive channels (in cp = 0.5, 1.0, 2.0), and a neutral channel (in cp = 0) are discussed. The results of the chain dimension and the energy show that Z 0 = 1.0 is an important case to distinguish the types of the channels. For the strong attractive channel, more contacts form during the process of translocation. It is also found that an external force is needed to drive the chain outside of the channel with the strong attraction. While for the neutral, the repulsive, and the weak attractive channels, the translocation is spontaneous. (interdisciplinary physics and related areas of science and technology)

  9. Chloride intracellular channel 1 identified using proteomic analysis plays an important role in the radiosensitivity of HEp-2 cells via reactive oxygen species production.

    Science.gov (United States)

    Kim, Jae-Sung; Chang, Jong Wook; Yun, Hong Shik; Yang, Kyung Mi; Hong, Eun-Hee; Kim, Dong Hyun; Um, Hong-Duck; Lee, Kee-Ho; Lee, Su-Jae; Hwang, Sang-Gu

    2010-07-01

    The nature of the molecules underlying the radioresistance phenotype of laryngeal cancer cells remains to be established. We initially generated radioresistant laryngeal cancer cell lines from human HEp-2 cells with fractionated radiation. These RR-HEp-2 cells and isolated clones displayed more radioresistant and anti-apoptotic phenotypes than parental HEp-2 cells after radiation. Characteristics of RR-Hep-2 cell lines were confirmed by upregulation of radioresistance-related genes, such as epidermal growth factor receptor, Hsp90, and Bcl-xl. Subsequently, we examined proteome changes between HEp-2 and RR-HEp-2 cells and identified 16 proteins showing significantly altered expression levels. Interestingly, protein expression of chloride intracellular channel 1 (CLIC1) was markedly suppressed in RR-HEp-2 cells, compared with non-irradiated control cells. Suppression of CLIC1 with an indanyloxyacetic acid-94 or small interfering RNA led to radioresistance in HEp-2 cells by suppressing the radiation-induced cellular ROS level. However, ectopic overexpression of CLIC1 induced radiosensitivity in RR-HEp-2 cells via induction of ROS level after radiation, suggesting that the protein acts as a positive regulator of ROS production. Our results collectively indicate that suppression of CLIC1 contributes to acquisition of the radioresistance phenotype of laryngeal cancer cells via inhibition of ROS production, implying that this protein is an important candidate molecule for radiotherapy in radioresistant laryngeal cancer cells.

  10. Photo-oxidation of cells generates long-lived intracellular protein peroxides

    DEFF Research Database (Denmark)

    Wright, Adam; Hawkins, Clare Louise; Davies, Michael Jonathan

    2003-01-01

    as a result of their abundance and high rate constants for reaction. In this study, we show that illumination of viable rose bengal-loaded THP-1 (human monocyte-like) cells with visible light gives rise to intracellular protein-derived peroxides. The peroxide yield increases with illumination time, requires....../mg cell protein, and account for up to approximately 15% of the O(2) consumed by the cells. Similar peroxides have been detected on isolated cellular proteins exposed to light in the presence of rose bengal and oxygen. After cessation of illumination, cellular protein peroxide levels decrease with t(1....../2) about 4 h at 37 degrees C. Decomposition of protein peroxides formed within cells, or on isolated cellular proteins, by metal ions gives rise to radicals as detected by EPR spin trapping. These studies demonstrate that exposure of intact cells to visible light in the presence of a sensitizer leads...

  11. p53 increases intra-cellular calcium release by transcriptional regulation of calcium channel TRPC6 in GaQ3-treated cancer cells.

    Directory of Open Access Journals (Sweden)

    Esha Madan

    Full Text Available p53 and calcium signaling are inter-dependent and are known to show both synergistic and antagonistic effects on each other in the cellular environment. However, no molecular mechanism or cellular pathway is known which shows direct regulation between these important cellular signaling molecules. Here we have shown that in cancer cells treated with anti-neoplastic drug GaQ3, p53, there is an increase in intracellular calcium levels by transcriptional regulation of a novel calcium channel gene TRPC6. p53 directly binds to a 22 bp response element in the TRPC6 gene promoter and increase its mRNA and protein expression. Over-expression of TRPC6 results in calcium-dependent apoptotic death and activation of apoptotic genes in a variety of cancer cells. This research work shows that p53 and its transcriptional activity is critical in regulation of calcium signaling and an increase in the intracellular calcium level might be one of the anti-cancer strategies to induce apoptosis in cancer cells.

  12. p53 increases intra-cellular calcium release by transcriptional regulation of calcium channel TRPC6 in GaQ3-treated cancer cells.

    Science.gov (United States)

    Madan, Esha; Gogna, Rajan; Keppler, Bernhard; Pati, Uttam

    2013-01-01

    p53 and calcium signaling are inter-dependent and are known to show both synergistic and antagonistic effects on each other in the cellular environment. However, no molecular mechanism or cellular pathway is known which shows direct regulation between these important cellular signaling molecules. Here we have shown that in cancer cells treated with anti-neoplastic drug GaQ3, p53, there is an increase in intracellular calcium levels by transcriptional regulation of a novel calcium channel gene TRPC6. p53 directly binds to a 22 bp response element in the TRPC6 gene promoter and increase its mRNA and protein expression. Over-expression of TRPC6 results in calcium-dependent apoptotic death and activation of apoptotic genes in a variety of cancer cells. This research work shows that p53 and its transcriptional activity is critical in regulation of calcium signaling and an increase in the intracellular calcium level might be one of the anti-cancer strategies to induce apoptosis in cancer cells.

  13. Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages.

    Science.gov (United States)

    Liang, Xue-hai; Sun, Hong; Shen, Wen; Crooke, Stanley T

    2015-03-11

    Although the RNase H-dependent mechanism of inhibition of gene expression by chemically modified antisense oligonucleotides (ASOs) has been well characterized, little is known about the interactions between ASOs and intracellular proteins that may alter cellular localization and/or potency of ASOs. Here, we report the identification of 56 intracellular ASO-binding proteins using multi-step affinity selection approaches. Many of the tested proteins had no significant effect on ASO activity; however, some proteins, including La/SSB, NPM1, ANXA2, VARS and PC4, appeared to enhance ASO activities, likely through mechanisms related to subcellular distribution. VARS and ANXA2 co-localized with ASOs in endocytic organelles, and reduction in the level of VARS altered lysosome/ASO localization patterns, implying that these proteins may facilitate ASO release from the endocytic pathway. Depletion of La and NPM1 reduced nuclear ASO levels, suggesting potential roles in ASO nuclear accumulation. On the other hand, Ku70 and Ku80 proteins inhibited ASO activity, most likely by competition with RNase H1 for ASO/RNA duplex binding. Our results demonstrate that phosphorothioate-modified ASOs bind a set of cellular proteins that affect ASO activity via different mechanisms. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Intracellular localization and interaction of mRNA binding proteins as detected by FRET.

    Science.gov (United States)

    David Gerecht, Pamela S; Taylor, Molly A; Port, J David

    2010-09-15

    A number of RNA binding proteins (BPs) bind to A+U rich elements (AREs), commonly present within 3'UTRs of highly regulated RNAs. Individual RNA-BPs proteins can modulate RNA stability, RNA localization, and/or translational efficiency. Although biochemical studies have demonstrated selectivity of ARE-BPs for individual RNAs, less certain is the in vivo composition of RNA-BP multiprotein complexes and how their composition is affected by signaling events and intracellular localization. Using FRET, we previously demonstrated that two ARE-BPs, HuR and AUF1, form stable homomeric and heteromeric associations in the nucleus and cytoplasm. In the current study, we use immuno-FRET of endogenous proteins to examine the intracellular localization and interactions of HuR and AUF1 as well as KSRP, TIA-1, and Hedls. These results were compared to those obtained with their exogenously expressed, fluorescently labeled counterparts. All ARE-BPs examined were found to colocalize and to form stable associations with selected other RNA-BPs in one or more cellular locations variably including the nucleus, cytoplasm (in general), or in stress granules or P bodies. Interestingly, FRET based interaction of the translational suppressor, TIA-1, and the decapping protein, Hedls, was found to occur at the interface of stress granules and P bodies, dynamic sites of intracellular RNA storage and/or turnover. To explore the physical interactions of RNA-BPs with ARE containing RNAs, in vitro transcribed Cy3-labeled RNA was transfected into cells. Interestingly, Cy3-RNA was found to coalesce in P body like punctate structures and, by FRET, was found to interact with the RNA decapping proteins, Hedls and Dcp1. Biochemical methodologies, such as co-immunoprecipitation, and cell biological approaches such as standard confocal microscopy are useful in demonstrating the possibility of proteins and/or proteins and RNAs interacting. However, as demonstrated herein, colocalization of proteins and

  15. Intracellular Protein Delivery and Gene Transfection by Electroporation Using a Microneedle Electrode Array

    Science.gov (United States)

    Choi, Seong-O; Kim, Yeu-Chun; Lee, Jeong Woo; Park, Jung-Hwan

    2012-01-01

    The impact of many biopharmaceuticals, including protein- and gene-based therapies, has been limited by the need for better methods of delivery into cells within tissues. Here, we present intracellular delivery of molecules and transfection with plasmid DNA by electroporation using a novel microneedle electrode array designed for targeted treatment of skin and other tissue surfaces. The microneedle array is molded out of polylactic acid. Electrodes and circuitry required for electroporation are applied to the microneedle array surface by a new metal-transfer micromolding method. The microneedle array maintains mechanical integrity after insertion into pig cadaver skin and is able to electroporate human prostate cancer cells in vitro. Quantitative measurements show that increasing electroporation pulse voltage increases uptake efficiency of calcein and bovine serum albumin, whereas increasing pulse length has lesser effects over the range studied. Uptake of molecules by up to 50 % of cells and transfection of 12 % of cells with a gene for green fluorescent protein is demonstrated at high cell viability. We conclude that the microneedle electrode array is able to electroporate cells, resulting in intracellular uptake of molecules, and has potential applications to improve intracellular delivery of proteins, DNA and other biopharmaceuticals. PMID:22328093

  16. Effect of serum proteins on polystyrene nanoparticle uptake and intracellular trafficking in endothelial cells

    International Nuclear Information System (INIS)

    Guarnieri, Daniela; Guaccio, Angela; Fusco, Sabato; Netti, Paolo A.

    2011-01-01

    The physico-chemical properties of nanoparticles (NPs), such as small dimensions, surface charge and surface functionalization, control their capability to interact with cells and, in particular, with sub-cellular components. This interaction can be also influenced by the adsorption of molecules present in biological fluids, like blood, on NP surface. Here, we analysed the effect of serum proteins on 49 and 100 nm red fluorescent polystyrene NP uptake in porcine aortic endothelial (PAE) cells, as a model for vascular transport. To this aim, NP uptake kinetic, endocytic pathway and intracellular trafficking were studied by monitoring NPs inside cells through confocal microscopy and multiple particle tracking (MPT). We demonstrated that NPs are rapidly internalized by cells in serum-free (SF) medium, according to a saturation kinetic. Conversely, in 10% foetal bovine serum-enriched (SE) medium, NP uptake rate results drastically reduced. Moreover, NP internalization depends on an active endocytic mechanism that does not involve clathrin- and caveolae-mediated vesicular transport, in both SE and SF media. Furthermore, MPT data indicate that NP intracellular trafficking is unaffected by protein presence. Indeed, approximately 50–60% of internalized NPs is characterized by a sub-diffusive behaviour, whereas the remaining fraction shows an active motion. These findings demonstrate that the unspecific protein adsorption on NP surface can affect cellular uptake in terms of internalization kinetics, but it is not effective in controlling active and cellular-mediated uptake mechanisms of NPs and their intracellular routes.

  17. Single channel analysis of membrane proteins in artificial bilayer membranes.

    Science.gov (United States)

    Bartsch, Philipp; Harsman, Anke; Wagner, Richard

    2013-01-01

    The planar lipid bilayer technique is a powerful experimental approach for electrical single channel recordings of pore-forming membrane proteins in a chemically well-defined and easily modifiable environment. Here we provide a general survey of the basic materials and procedures required to set up a robust bilayer system and perform electrophysiological single channel recordings of reconstituted proteins suitable for the in-depth characterization of their functional properties.

  18. Comparison of intracellular localization of Nubp1 and Nubp2 using GFP fusion proteins.

    Science.gov (United States)

    Okuno, Takashi; Yamabayashi, Hiroko; Kogure, Kentaro

    2010-03-01

    Nubp1 (also known as Nbp35) and Nubp2 (also known as Cfd1) proteins are known to be responsible for regulating centrosome duplication in mouse and ribosome biogenesis in yeast. Nubp proteins contribute to diverse physiological functions. It is thought that Nubp1 and Nubp2 proteins interact with each other and regulate their functions. However, little is known about the intracellular localization of Nubp proteins. In this study, we compared the intracellular localization of human Nubp1 and Nubp2 by fusing these proteins with green fluorescent protein (GFP) in HeLa cells. The nuclear transfer of Nubp1-GFP, where GFP was fused to the C-terminus, was not observed. However, GFP-Nubp1, where GFP was fused to the N-terminus, did accumulate in the nucleus. In addition, GFP-modification at the N-terminal of Nubp2 induced nuclear transformation. Our data suggest that the C-terminal region of Nubp1 is important for nuclear transfer and the N-terminal of Nubp2 contributes to the morphology of the nucleus.

  19. Molecular and Physiological Analysis of a Thylakoid K+ Channel Protein.

    Science.gov (United States)

    Fang, Z.; Mi, F.; Berkowitz, G. A.

    1995-08-01

    Transport studies identified a K+ channel protein in preparations of purified spinach (Spinacea oleracea) thylakoid membrane. This protein was solubilized from native membranes and reconstituted into artificial proteoliposomes with maintenance of functional integrity. A 33-kD thylakoid polypeptide was identified as a putative component of this thylakoid protein. This identification was made using an antibody raised against a synthetic peptide representing a highly conserved region of K+ channel proteins. K+ channel activity co-migrated with the immunoreactive 33-kD polypeptide when solubilized thylakoid membrane protein was fractionated on a Suc density gradient. The antibody was used to immunoprecipitate the 33-kD polypeptide. Physiological function of this thylakoid membrane protein was elucidated by measuring photosynthetic electron transport of thylakoid preparations in the presence and absence of a K+ channel blocker. Results indicated that K+ efflux from the thylakoid lumen through this channel protein is required for the optimization of photosynthetic capacity. The effect this protein has on photosynthetic capacity is likely due to the requirement for K+ efflux from the thylakoid lumen to charge-balance light-induced proton pumping across this membrane.

  20. Amoxicillin haptenates intracellular proteins that can be transported in exosomes to target cells.

    Science.gov (United States)

    Sánchez-Gómez, F J; González-Morena, J M; Vida, Y; Pérez-Inestrosa, E; Blanca, M; Torres, M J; Pérez-Sala, D

    2017-03-01

    Allergic reactions to β-lactams are among the most frequent causes of drug allergy and constitute an important clinical problem. Drug covalent binding to endogenous proteins (haptenation) is thought to be required for activation of the immune system. Nevertheless, neither the nature nor the role of the drug protein targets involved in this process is fully understood. Here, we aim to identify novel intracellular targets for haptenation by amoxicillin (AX) and their cellular fate. We have treated B lymphocytes with either AX or a biotinylated analog (AX-B). The identification of protein targets for haptenation by AX has been approached by mass spectrometry and immunoaffinity techniques. In addition, intercellular communication mediated by the delivery of vesicles loaded with AX-B-protein adducts has been explored by microscopy techniques. We have observed a complex pattern of AX-haptenated proteins. Several novel targets for haptenation by AX in B lymphocytes have been identified. AX-haptenated proteins were detected in cell lysates and extracellularly, either as soluble proteins or in lymphocyte-derived extracellular vesicles. Interestingly, exosomes from AX-B-treated cells showed a positive biotin signal in electron microscopy. Moreover, they were internalized by endothelial cells, thus supporting their involvement in intercellular transfer of haptenated proteins. These results represent the first identification of AX-mediated haptenation of intracellular proteins. Moreover, they show that exosomes can constitute a novel vehicle for haptenated proteins, and raise the hypothesis that they could provide antigens for activation of the immune system during the allergic response. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Intracellular localization of Equine herpesvirus type 1 tegument protein VP22.

    Science.gov (United States)

    Okada, Ayaka; Kodaira, Akari; Hanyu, Sachiko; Izume, Satoko; Ohya, Kenji; Fukushi, Hideto

    2014-11-04

    Intracellular localization of Equine herpesvirus type 1 (EHV-1) tegument protein VP22 was examined by using a plasmid that expressed VP22 fused with an enhanced green fluorescent protein (EGFP). Also a recombinant EHV-1 expressing VP22 fused with a red fluorescent protein (mCherry) was constructed to observe the localization of VP22 in infected cells. When EGFP-fused VP22 was overexpressed in the cells, VP22 localized in the cytoplasm and nucleus. Live cell imaging suggested that the fluorescently tagged VP22 also localized in the cytoplasm and nucleus. These results show that VP22 localizes in the cytoplasm and nucleus independently of other viral proteins. Experiments with truncation mutants of pEGFP-VP22 suggested that 154-188 aa might be the nuclear localization signal of EHV-1 VP22. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Intracellular polyamine pools, oligopeptide-binding protein A expression, and resistance to aminoglycosides in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Maria BR Acosta

    2005-11-01

    Full Text Available The role of intracellular free polyamine (putrescine and spermidine pools in multiple resistance to aminoglycoside antibiotics was investigated among in vitro selected kanamycin-resistant Escherichia coli J53 mutants expressing diminished oligopeptide-binding protein (OppA levels and/or defective ornithine decarboxylase (ODC activity. The results suggest that diminished OppA content, but not defective ODC activity expression, increased the relative concentration of free spermidine as compared to the wild type strain. Moreover, by adding exogenous polyamines or polyamine synthesis inhibitors to cultures with different mutant strains, a direct relationship between the intracellular OppA levels and resistance to kanamycin was revealed. Collectively these results further suggest a complex relation among OppA expression, aminoglycoside resistance and polyamine metabolism.

  3. Single proteins that serve linked functions in intracellular and extracellular microenvironments

    Energy Technology Data Exchange (ETDEWEB)

    Radisky, Derek C.; Stallings-Mann, Melody; Hirai, Yohei; Bissell, Mina J.

    2009-06-03

    Maintenance of organ homeostasis and control of appropriate response to environmental alterations requires intimate coordination of cellular function and tissue organization. An important component of this coordination may be provided by proteins that can serve distinct, but linked, functions on both sides of the plasma membrane. Here we present a novel hypothesis in which non-classical secretion can provide a mechanism through which single proteins can integrate complex tissue functions. Single genes can exert a complex, dynamic influence through a number of different processes that act to multiply the function of the gene product(s). Alternative splicing can create many different transcripts that encode proteins of diverse, even antagonistic, function from a single gene. Posttranslational modifications can alter the stability, activity, localization, and even basic function of proteins. A protein can exist in different subcellular localizations. More recently, it has become clear that single proteins can function both inside and outside the cell. These proteins often lack defined secretory signal sequences, and transit the plasma membrane by mechanisms separate from the classical ER/Golgi secretory process. When examples of such proteins are examined individually, the multifunctionality and lack of a signal sequence are puzzling - why should a protein with a well known function in one context function in such a distinct fashion in another? We propose that one reason for a single protein to perform intracellular and extracellular roles is to coordinate organization and maintenance of a global tissue function. Here, we describe in detail three specific examples of proteins that act in this fashion, outlining their specific functions in the extracellular space and in the intracellular space, and we discuss how these functions may be linked. We present epimorphin/syntaxin-2, which may coordinate morphogenesis of secretory organs (as epimorphin) with control of

  4. Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta.

    Science.gov (United States)

    Cabral, Wayne A; Ishikawa, Masaki; Garten, Matthias; Makareeva, Elena N; Sargent, Brandi M; Weis, MaryAnn; Barnes, Aileen M; Webb, Emma A; Shaw, Nicholas J; Ala-Kokko, Leena; Lacbawan, Felicitas L; Högler, Wolfgang; Leikin, Sergey; Blank, Paul S; Zimmerberg, Joshua; Eyre, David R; Yamada, Yoshihiko; Marini, Joan C

    2016-07-01

    Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50-70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes.

  5. Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta.

    Directory of Open Access Journals (Sweden)

    Wayne A Cabral

    2016-07-01

    Full Text Available Recessive osteogenesis imperfecta (OI is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50-70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes.

  6. Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta

    Science.gov (United States)

    Cabral, Wayne A.; Ishikawa, Masaki; Garten, Matthias; Makareeva, Elena N.; Sargent, Brandi M.; Weis, MaryAnn; Barnes, Aileen M.; Webb, Emma A.; Shaw, Nicholas J.; Ala-Kokko, Leena; Lacbawan, Felicitas L.; Högler, Wolfgang; Leikin, Sergey; Blank, Paul S.; Zimmerberg, Joshua; Eyre, David R.; Yamada, Yoshihiko; Marini, Joan C.

    2016-01-01

    Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50–70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes. PMID:27441836

  7. Intracellular hepatitis C modeling predicts infection dynamics and viral protein mechanisms.

    Science.gov (United States)

    Aunins, Thomas R; Marsh, Katherine A; Subramanya, Gitanjali; Uprichard, Susan L; Perelson, Alan S; Chatterjee, Anushree

    2018-03-21

    Hepatitis C virus infection is a global health problem, with nearly 2 million new infections occurring every year and up to 85% of these becoming chronic infections that pose serious long-term health risks. To effectively reduce the prevalence of HCV infection and associated diseases, it is important to understand the intracellular dynamics of the viral lifecycle. Here, we present a detailed mathematical model that represents the full hepatitis C lifecycle. It is the first full HCV model to be fit to acute intracellular infection data and the first to explore the functions of distinct viral proteins, probing multiple hypotheses of cis - and trans -acting mechanisms to provide insights for drug targeting. Model parameters were derived from the literature, experiments, and fitting to experimental intracellular viral RNA, extracellular viral titer, and HCV core and NS3 protein kinetic data from viral inoculation to steady-state. Our model predicts faster rates for protein translation and polyprotein cleavage than previous replicon models and demonstrates that the processes of translation and synthesis of viral RNA have the most influence on the levels of the species we tracked in experiments. Overall, our experimental data and the resulting mathematical infection model reveal information about the regulation of core protein during infection, produce specific insights into the roles of the viral core, NS5A, and NS5B proteins, and demonstrate the sensitivities of viral proteins and RNA to distinct reactions within the lifecycle. IMPORTANCE We have designed a model for the full lifecycle of hepatitis C virus. Past efforts have largely focused on modeling hepatitis C replicon systems, in which transfected subgenomic HCV RNA maintains autonomous replication in the absence of virion production or spread. We started with the general structure of these previous replicon models and expanded to create a model that incorporates the full virus lifecycle as well as additional

  8. The Intracellular Destiny of the Protein Corona: A Study on its Cellular Internalization and Evolution.

    Science.gov (United States)

    Bertoli, Filippo; Garry, David; Monopoli, Marco P; Salvati, Anna; Dawson, Kenneth A

    2016-11-22

    It has been well established that the early stages of nanoparticle-cell interactions are governed, at least in part, by the layer of proteins and other biomolecules adsorbed and slowly exchanged with the surrounding biological media (biomolecular corona). Subsequent to membrane interactions, nanoparticles are typically internalized into the cell and trafficked along defined pathways such as, in many cases, the endolysosomal pathway. Indeed, if the original corona is partially retained on the nanoparticle surface, the biomolecules in this layer may play an important role in determining subsequent cellular processing. In this work, using a combination of organelle separation and fluorescence labeling of the initial extracellular corona, we clarify its intracellular evolution as nanoparticles travel within the cell. We show that specific proteins present in the original protein corona are retained on the nanoparticles until they accumulate in lysosomes, and, once there, they are degraded. We also report on how different bare surfaces (amino and carboxyl modified) affect the details of this evolution. One overarching discovery is that the same serum proteins can exhibit different intracellular processing when carried inside cells by nanoparticles, as components of their corona, compared to what is observed when they are transported freely from the extracellular medium.

  9. The DNAJB6 and DNAJB8 protein chaperones prevent intracellular aggregation of polyglutamine peptides.

    Science.gov (United States)

    Gillis, Judith; Schipper-Krom, Sabine; Juenemann, Katrin; Gruber, Anna; Coolen, Silvia; van den Nieuwendijk, Rian; van Veen, Henk; Overkleeft, Hermen; Goedhart, Joachim; Kampinga, Harm H; Reits, Eric A

    2013-06-14

    Fragments of proteins containing an expanded polyglutamine (polyQ) tract are thought to initiate aggregation and toxicity in at least nine neurodegenerative diseases, including Huntington's disease. Because proteasomes appear unable to digest the polyQ tract, which can initiate intracellular protein aggregation, preventing polyQ peptide aggregation by chaperones should greatly improve polyQ clearance and prevent aggregate formation. Here we expressed polyQ peptides in cells and show that their intracellular aggregation is prevented by DNAJB6 and DNAJB8, members of the DNAJ (Hsp40) chaperone family. In contrast, HSPA/Hsp70 and DNAJB1, also members of the DNAJ chaperone family, did not prevent peptide-initiated aggregation. Intriguingly, DNAJB6 and DNAJB8 also affected the soluble levels of polyQ peptides, indicating that DNAJB6 and DNAJB8 inhibit polyQ peptide aggregation directly. Together with recent data showing that purified DNAJB6 can suppress fibrillation of polyQ peptides far more efficiently than polyQ expanded protein fragments in vitro, we conclude that the mechanism of DNAJB6 and DNAJB8 is suppression of polyQ protein aggregation by directly binding the polyQ tract.

  10. Production of Candida utilis Biomass and Intracellular Protein Content: Effect of Agitation Speed and Aeration Rate

    Directory of Open Access Journals (Sweden)

    Rosma, A.

    2006-01-01

    Full Text Available The effects of agitation speed and aeration rate on the Candida utilis biomass and the intracellular protein content were investigated in this study. C. utilis inoculum of 10^6 cells/mL (7.8 % v/v was cultured in 1.5 L pineapple waste medium (3 % Brix in a 2-L fermentor for 30 h at 30 °C. Agitation speed and aeration rate have significant effects on the dissolved oxygen concentration, which in turn affect the cell growth and the intracellular protein content. The agitation speed of 100, 300, 500, 700 and 900 rpm was employed. The highest yield of protein content (1.2 g/L media and total biomass (7.8 g/L media were resulted from yeast cultivation with agitation speed of 900 rpm. Thus, the effects of aeration rate (0.5, 1.0, 2.0 and 3.0 L/min were studied at agitation speed of 900 rpm. A maximum yield of protein content (1.6 g/L media and biomass (9.5 g/L media were attained at aeration rate of 2.0 L/min.

  11. Cooperative regulation by G proteins and Na(+) of neuronal GIRK2 K(+) channels.

    Science.gov (United States)

    Wang, Weiwei; Touhara, Kouki K; Weir, Keiko; Bean, Bruce P; MacKinnon, Roderick

    2016-04-13

    G protein gated inward rectifier K(+) (GIRK) channels open and thereby silence cellular electrical activity when inhibitory G protein coupled receptors (GPCRs) are stimulated. Here we describe an assay to measure neuronal GIRK2 activity as a function of membrane-anchored G protein concentration. Using this assay we show that four Gβγ subunits bind cooperatively to open GIRK2, and that intracellular Na(+) - which enters neurons during action potentials - further amplifies opening mostly by increasing Gβγ affinity. A Na(+) amplification function is characterized and used to estimate the concentration of Gβγ subunits that appear in the membrane of mouse dopamine neurons when GABAB receptors are stimulated. We conclude that GIRK2, through its dual responsiveness to Gβγ and Na(+), mediates a form of neuronal inhibition that is amplifiable in the setting of excess electrical activity.

  12. Regulation of biosynthesis and intracellular localization of rice and tobacco homologues of nucleosome assembly protein 1.

    Science.gov (United States)

    Dong, Aiwu; Zhu, Yan; Yu, Yu; Cao, Kaiming; Sun, Chongrong; Shen, Wen-Hui

    2003-02-01

    The nucleosome assembly protein 1 (NAP1) is considered to be a conserved histone chaperone, facilitating the assembly of nucleosomes in all eukaryotes. However, studies in yeast and animal cells also indicated that NAP1 proteins have diverse functions likely independent of nucleosome-assembly activity. Here, we describe the isolation and characterization of cDNAs encoding NAP1-like proteins from the monocotyledon rice ( Oryza sativa L.) and the dicotyledon tobacco ( Nicotiana tabacum L.). Northern-blot analysis demonstrated that the two rice NAP1-like genes are predominantly expressed in stem tissues such as root and shoot apical meristems as well as in young flowers. During the cell cycle, all four tobacco NAP1-like genes are highly expressed, with one of them showing a slightly increased expression at the G1/S transition. These results are consistent with a role for plant NAP1-like proteins in cell division. In vitro binding assays revealed that different NAP1-like proteins bind, with distinct relative binding strengths, to different classes of histone. Intracellular localization analyses showed that some NAP1-like proteins could be targeted into the nucleus whereas others are exclusively cytoplasm-localized. It is thus likely that different plant NAP1-like proteins have distinct functions in vivo. Plant NAP1-like proteins were observed to concentrate around the metaphase plate and in the phragmoplast, suggesting a role in mitotic events and cytokinesis.

  13. Integrating Protein Engineering and Bioorthogonal Click Conjugation for Extracellular Vesicle Modulation and Intracellular Delivery.

    Directory of Open Access Journals (Sweden)

    Ming Wang

    Full Text Available Exosomes are small, cell-secreted vesicles that transfer proteins and genetic information between cells. This intercellular transmission regulates many physiological and pathological processes. Therefore, exosomes have emerged as novel biomarkers for disease diagnosis and as nanocarriers for drug delivery. Here, we report an easy-to-adapt and highly versatile methodology to modulate exosome composition and conjugate exosomes for intracellular delivery. Our strategy combines the metabolic labeling of newly synthesized proteins or glycan/glycoproteins of exosome-secreting cells with active azides and bioorthogonal click conjugation to modify and functionalize the exosomes. The azide-integrated can be conjugated to a variety of small molecules and proteins and can efficiently deliver conjugates into cells. The metabolic engineering of exosomes diversifies the chemistry of exosomes and expands the functions that can be introduced into exosomes, providing novel, powerful tools to study the roles of exosomes in biology and expand the biomedical potential of exosomes.

  14. Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication.

    Science.gov (United States)

    Brödel, Andreas K; Jaramillo, Alfonso; Isalan, Mark

    2017-09-01

    Directed evolution is a powerful tool to improve the characteristics of biomolecules. Here we present a protocol for the intracellular evolution of proteins with distinct differences and advantages in comparison with established techniques. These include the ability to select for a particular function from a library of protein variants inside cells, minimizing undesired coevolution and propagation of nonfunctional library members, as well as allowing positive and negative selection logics using basally active promoters. A typical evolution experiment comprises the following stages: (i) preparation of a combinatorial M13 phagemid (PM) library expressing variants of the gene of interest (GOI) and preparation of the Escherichia coli host cells; (ii) multiple rounds of an intracellular selection process toward a desired activity; and (iii) the characterization of the evolved target proteins. The system has been developed for the selection of new orthogonal transcription factors (TFs) but is capable of evolving any gene-or gene circuit function-that can be linked to conditional M13 phage replication. Here we demonstrate our approach using as an example the directed evolution of the bacteriophage λ cI TF against two synthetic bidirectional promoters. The evolved TF variants enable simultaneous activation and repression against their engineered promoters and do not cross-react with the wild-type promoter, thus ensuring orthogonality. This protocol requires no special equipment, allowing synthetic biologists and general users to evolve improved biomolecules within ∼7 weeks.

  15. Bisindoylmaleimide I suppresses adipocyte differentiation through stabilization of intracellular β-catenin protein

    International Nuclear Information System (INIS)

    Cho, Munju; Park, Seoyoung; Gwak, Jungsug; Kim, Dong-Eun; Yea, Sung Su; Shin, Jae-Gook; Oh, Sangtaek

    2008-01-01

    The Wnt/β-catenin signaling pathway plays important roles in cell differentiation. Activation of this pathway, likely by Wnt-10b, has been shown to inhibit adipogenesis in cultured 3T3-L1 preadipocytes and mice. Here we revealed that bisindoylmaleimide I (BIM), which is widely used as a specific inhibitor of protein kinase C (PKC), inhibits adipocyte differentiation through activation of the Wnt/β-catenin signaling pathway. BIM increased β-catenin responsive transcription (CRT) and up-regulated intracellular β-catenin levels in HEK293 cells and 3T3-L1 preadipocytes. BIM significantly decreased intracellular lipid accumulation and reduced expression of important adipocyte marker genes including peroxisome-proliferator-activated receptor γ (PPARγ) and CAATT enhancer-binding protein α (C/EBPα) in 3T3-L1 preadipocytes. Taken together, our findings indicate that BIM inhibits adipogenesis by increasing the stability of β-catenin protein in 3T3-L1 preadipocyte cells

  16. Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

    Science.gov (United States)

    Unzueta, Ugutz; Céspedes, María Virtudes; Ferrer-Miralles, Neus; Casanova, Isolda; Cedano, Juan; Corchero, José Luis; Domingo-Espín, Joan; Villaverde, Antonio; Mangues, Ramón; Vázquez, Esther

    2012-01-01

    Background Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4) is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing. Results Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer. Conclusion Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles, and imaging agents. PMID:22923991

  17. Intracellular localization of adeno-associated viral proteins expressed in insect cells.

    Science.gov (United States)

    Gallo-Ramírez, Lilí E; Ramírez, Octavio T; Palomares, Laura A

    2011-01-01

    Production of vectors derived from adeno-associated virus (AAVv) in insect cells represents a feasible option for large-scale applications. However, transducing particles yields obtained in this system are low compared with total capsid yields, suggesting the presence of genome encapsidation bottlenecks. Three components are required for AAVv production: viral capsid proteins (VP), the recombinant AAV genome, and Rep proteins for AAV genome replication and encapsidation. Little is known about the interaction between the three components in insect cells, which have intracellular conditions different to those in mammalian cells. In this work, the localization of AAV proteins in insect cells was assessed for the first time with the purpose of finding potential limiting factors. Unassembled VP were located either in the cytoplasm or in the nucleus. Their transport into the nucleus was dependent on protein concentration. Empty capsids were located in defined subnuclear compartments. Rep proteins expressed individually were efficiently translocated into the nucleus. Their intranuclear distribution was not uniform and differed from VP distribution. While Rep52 distribution and expression levels were not affected by AAV genomes or VP, Rep78 distribution and stability changed during coexpression. Expression of all AAV components modified capsid intranuclear distribution, and assembled VP were found in vesicles located in the nuclear periphery. Such vesicles were related to baculovirus infection, highlighting its role in AAVv production in insect cells. The results obtained in this work suggest that the intracellular distribution of AAV proteins allows their interaction and does not limit vector production in insect cells. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

  18. Deep Ultraviolet Mapping of Intracellular Protein and Nucleic Acid in Femtograms per Pixel

    OpenAIRE

    Cheung, Man C.; Evans, James G.; McKenna, Brian; Ehrlich, Daniel J.

    2011-01-01

    By using imaging spectrophotometry with paired images in the 200- to 280-nm wavelength range, we have directly mapped intracellular nucleic acid and protein distributions across a population of Chinese hamster ovary (CHO-K1) cells. A broadband 100× objective with a numerical aperture of 1.2NA (glycerin immersion) and a novel laser-induced-plasma point source generated high-contrast images with short (~100 ms) exposures and a lateral resolution nearing 200 nm that easily resolves internal orga...

  19. Aroclor 1254, a developmental neurotoxicant, alters energy metabolism- and intracellular signaling-associated protein networks in rat cerebellum and hippocampus

    Energy Technology Data Exchange (ETDEWEB)

    Kodavanti, Prasada Rao S., E-mail: kodavanti.prasada@epa.gov [Neurotoxicology Branch, NHEERL, ORD, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina (United States); Osorio, Cristina [Systems Proteomics Center, University of North Carolina at Chapel Hill, North Carolina (United States); Program on Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, North Carolina (United States); Royland, Joyce E.; Ramabhadran, Ram [Genetic and Cellular Toxicology Branch, NHEERL, ORD, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina (United States); Alzate, Oscar [Department of Cellular and Developmental Biology, University of North Carolina at Chapel Hill, North Carolina (United States); Systems Proteomics Center, University of North Carolina at Chapel Hill, North Carolina (United States); Program on Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, North Carolina (United States)

    2011-11-15

    The vast literature on the mode of action of polychlorinated biphenyls (PCBs) indicates that PCBs are a unique model for understanding the mechanisms of toxicity of environmental mixtures of persistent chemicals. PCBs have been shown to adversely affect psychomotor function and learning and memory in humans. Although the molecular mechanisms for PCB effects are unclear, several studies indicate that the disruption of Ca{sup 2+}-mediated signal transduction plays significant roles in PCB-induced developmental neurotoxicity. Culminating events in signal transduction pathways include the regulation of gene and protein expression, which affects the growth and function of the nervous system. Our previous studies showed changes in gene expression related to signal transduction and neuronal growth. In this study, protein expression following developmental exposure to PCB is examined. Pregnant rats (Long Evans) were dosed with 0.0 or 6.0 mg/kg/day of Aroclor-1254 from gestation day 6 through postnatal day (PND) 21, and the cerebellum and hippocampus from PND14 animals were analyzed to determine Aroclor 1254-induced differential protein expression. Two proteins were found to be differentially expressed in the cerebellum following PCB exposure while 18 proteins were differentially expressed in the hippocampus. These proteins are related to energy metabolism in mitochondria (ATP synthase, sub unit {beta} (ATP5B), creatine kinase, and malate dehydrogenase), calcium signaling (voltage-dependent anion-selective channel protein 1 (VDAC1) and ryanodine receptor type II (RyR2)), and growth of the nervous system (dihydropyrimidinase-related protein 4 (DPYSL4), valosin-containing protein (VCP)). Results suggest that Aroclor 1254-like persistent chemicals may alter energy metabolism and intracellular signaling, which might result in developmental neurotoxicity. -- Highlights: Black-Right-Pointing-Pointer We performed brain proteomic analysis of rats exposed to the neurotoxicant

  20. Antagonists of the TMEM16A calcium-activated chloride channel modulate airway smooth muscle tone and intracellular calcium.

    Science.gov (United States)

    Danielsson, Jennifer; Perez-Zoghbi, Jose; Bernstein, Kyra; Barajas, Matthew B; Zhang, Yi; Kumar, Satish; Sharma, Pawan K; Gallos, George; Emala, Charles W

    2015-09-01

    Perioperative bronchospasm refractory to β agonists continues to challenge anesthesiologists and intensivists. The TMEM16A calcium-activated chloride channel modulates airway smooth muscle (ASM) contraction. The authors hypothesized that TMEM16A antagonists would relax ASM contraction by modulating membrane potential and calcium flux. Human ASM, guinea pig tracheal rings, or mouse peripheral airways were contracted with acetylcholine or leukotriene D4 and then treated with the TMEM16A antagonists: benzbromarone, T16Ainh-A01, N-((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid, or B25. In separate studies, guinea pig tracheal rings were contracted with acetylcholine and then exposed to increasing concentrations of isoproterenol (0.01 nM to 10 μM) ± benzbromarone. Plasma membrane potential and intracellular calcium concentrations were measured in human ASM cells. Benzbromarone was the most potent TMEM16A antagonist tested for relaxing an acetylcholine -induced contraction in guinea pig tracheal rings (n = 6). Further studies were carried out to investigate the clinical utility of benzbromarone. In human ASM, benzbromarone relaxed either an acetylcholine- or a leukotriene D4-induced contraction (n = 8). Benzbromarone was also effective in relaxing peripheral airways (n = 9) and potentiating relaxation by β agonists (n = 5 to 10). In cellular mechanistic studies, benzbromarone hyperpolarized human ASM cells (n = 9 to 12) and attenuated intracellular calcium flux from both the plasma membrane and the sarcoplasmic reticulum (n = 6 to 12). TMEM16A antagonists work synergistically with β agonists and through a novel pathway of interrupting ion flux at both the plasma membrane and sarcoplasmic reticulum to acutely relax human ASM.

  1. Deep Ultraviolet Mapping of Intracellular Protein and Nucleic Acid in Femtograms per Pixel

    Science.gov (United States)

    Cheung, Man C.; Evans, James G.; McKenna, Brian; Ehrlich, Daniel J.

    2011-01-01

    By using imaging spectrophotometry with paired images in the 200- to 280-nm wavelength range, we have directly mapped intracellular nucleic acid and protein distributions across a population of Chinese hamster ovary (CHO-K1) cells. A broadband 100× objective with a numerical aperture of 1.2NA (glycerin immersion) and a novel laser-induced-plasma point source generated high-contrast images with short (~100 ms) exposures and a lateral resolution nearing 200 nm that easily resolves internal organelles. In a population of 420 CHO-K1 cells and 477 nuclei, we found a G1 whole-cell nucleic acid peak at 26.6 pg, a nuclear-isolated total nucleic acid peak at 11.4 pg, and, as inferred by RNase treatment, a G1 total DNA mass of 7.4 pg. At the G1 peak we found a whole-cell protein mass of 95.6 pg, and a nuclear-isolated protein mass of 39.3 pg. An algorithm for protein quantification that senses peptide-bond (220-nm) absorbance was found to have a higher signal-to-noise ratio and to provide more reliable nucleic acid and protein determinations when compared to more classical 280-nm/260-nm algorithms when used for intracellular mass mapping. Using simultaneous imaging with common nuclear stains (Hoechst 33342, Syto-14, and Sytox Orange), we have compared staining patterns to deep-UV images of condensed chromatin and have confirmed bias of these common nuclear stains related to nuclear packaging. The approach allows absolute mass measurements with no special sample preparation or staining. It can be used in conjunction with normal fluorescence microscopy and with relatively modest modification of the microscope. PMID:21796773

  2. PIAS proteins are involved in the SUMO-1 modification, intracellular translocation and transcriptional repressive activity of RET finger protein

    International Nuclear Information System (INIS)

    Matsuura, Tetsuo; Shimono, Yohei; Kawai, Kumi; Murakami, Hideki; Urano, Takeshi; Niwa, Yasumasa; Goto, Hidemi; Takahashi, Masahide

    2005-01-01

    Ret finger protein (RFP) is a nuclear protein that is highly expressed in testis and in various tumor cell lines. RFP functions as a transcriptional repressor and associates with Enhancer of Polycomb 1 (EPC1), a member of the Polycomb group proteins, and Mi-2β, a main component of the nucleosome remodeling and deacetylase (NuRD) complex. We show that RFP binds with PIAS (protein inhibitor of activated STAT) proteins, PIAS1, PIAS3, PIASxα and PIASy at their carboxyl-terminal region and is covalently modified by SUMO-1 (sumoylation). PIAS proteins enhance the sumoylation of RFP in a dose-dependent manner and induce the translocation of RFP into nuclear bodies reminiscent of the PML bodies. In addition, co-expression of PIAS proteins or SUMO-1 strengthened the transcriptional repressive activity of RFP. Finally, our immunohistochemical results show that RFP, SUMO-1 and PIASy localize in a characteristic nuclear structure juxtaposed with the inner nuclear membrane (XY body) of primary spermatocytes in mouse testis. These results demonstrate that the intracellular location and the transcriptional activity of RFP are modified by PIAS proteins which possess SUMO E3 ligase activities and suggest that they may play a co-operative role in spermatogenesis

  3. Intracellular calcium levels determine differential modulation of allosteric interactions within G protein-coupled receptor heteromers.

    Science.gov (United States)

    Navarro, Gemma; Aguinaga, David; Moreno, Estefania; Hradsky, Johannes; Reddy, Pasham P; Cortés, Antoni; Mallol, Josefa; Casadó, Vicent; Mikhaylova, Marina; Kreutz, Michael R; Lluís, Carme; Canela, Enric I; McCormick, Peter J; Ferré, Sergi

    2014-11-20

    The pharmacological significance of the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer is well established and it is being considered as an important target for the treatment of Parkinson’s disease and other neuropsychiatric disorders. However, the physiological factors that control its distinctive biochemical properties are still unknown. We demonstrate that different intracellular Ca2+ levels exert a differential modulation of A2AR-D2R heteromer-mediated adenylyl-cyclase and MAPK signaling in striatal cells. This depends on the ability of low and high Ca2+ levels to promote a selective interaction of the heteromer with the neuronal Ca2+-binding proteins NCS-1 and calneuron-1, respectively. These Ca2+-binding proteins differentially modulate allosteric interactions within the A2AR-D2R heteromer, which constitutes a unique cellular device that integrates extracellular (adenosine and dopamine) and intracellular (Ca+2) signals to produce a specific functional response.

  4. Vitamin E and Phosphoinositides Regulate the Intracellular Localization of the Hepatic α-Tocopherol Transfer Protein.

    Science.gov (United States)

    Chung, Stacey; Ghelfi, Mikel; Atkinson, Jeffrey; Parker, Robert; Qian, Jinghui; Carlin, Cathleen; Manor, Danny

    2016-08-12

    α-Tocopherol (vitamin E) is an essential nutrient for all vertebrates. From the eight naturally occurring members of the vitamin E family, α-tocopherol is the most biologically active species and is selectively retained in tissues. The hepatic α-tocopherol transfer protein (TTP) preferentially selects dietary α-tocopherol and facilitates its transport through the hepatocyte and its secretion to the circulation. In doing so, TTP regulates body-wide levels of α-tocopherol. The mechanisms by which TTP facilitates α-tocopherol trafficking in hepatocytes are poorly understood. We found that the intracellular localization of TTP in hepatocytes is dynamic and responds to the presence of α-tocopherol. In the absence of the vitamin, TTP is localized to perinuclear vesicles that harbor CD71, transferrin, and Rab8, markers of the recycling endosomes. Upon treatment with α-tocopherol, TTP- and α-tocopherol-containing vesicles translocate to the plasma membrane, prior to secretion of the vitamin to the exterior of the cells. The change in TTP localization is specific to α-tocopherol and is time- and dose-dependent. The aberrant intracellular localization patterns of lipid binding-defective TTP mutants highlight the importance of protein-lipid interaction in the transport of α-tocopherol. These findings provide the basis for a proposed mechanistic model that describes TTP-facilitated trafficking of α-tocopherol through hepatocytes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. A variant of green fluorescent protein exclusively deposited to active intracellular inclusion bodies.

    Science.gov (United States)

    Raghunathan, Govindan; Munussami, Ganapathiraman; Moon, Hyojin; Paik, Hyun-jong; An, Seong Soo A; Kim, Yong-Sung; Kang, Sebyung; Lee, Sun-Gu

    2014-05-16

    Inclusion bodies (IBs) were generally considered to be inactive protein deposits and did not hold any attractive values in biotechnological applications. Recently, some IBs of recombinant proteins were confirmed to show their functional properties such as enzyme activities, fluorescence, etc. Such biologically active IBs are not commonly formed, but they have great potentials in the fields of biocatalysis, material science and nanotechnology. In this study, we characterized the IBs of DL4, a deletion variant of green fluorescent protein which forms active intracellular aggregates. The DL4 proteins expressed in Escherichia coli were exclusively deposited to IBs, and the IBs were estimated to be mostly composed of active proteins. The spectral properties and quantum yield of the DL4 variant in the active IBs were almost same with those of its native protein. Refolding and stability studies revealed that the deletion mutation in DL4 didn't affect the folding efficiency of the protein, but destabilized its structure. Analyses specific for amyloid-like structures informed that the inner architecture of DL4 IBs might be amorphous rather than well-organized. The diameter of fluorescent DL4 IBs could be decreased up to 100-200 nm by reducing the expression time of the protein in vivo. To our knowledge, DL4 is the first GFP variant that folds correctly but aggregates exclusively in vivo without any self-aggregating/assembling tags. The fluorescent DL4 IBs have potentials to be used as fluorescent biomaterials. This study also suggests that biologically active IBs can be achieved through engineering a target protein itself.

  6. Functional analysis of picornavirus 2B proteins: effects on calcium homeostasis and intracellular protein trafficking.

    NARCIS (Netherlands)

    Jong, A.S. de; Mattia, F.P. de; Dommelen, M.M.T.; Lanke, K.H.W.; Melchers, W.J.G.; Willems, P.H.G.M.; Kuppeveld, F.J.M. van

    2008-01-01

    The family Picornaviridae consists of a large group of plus-strand RNA viruses that share a similar genome organization. The nomenclature of the picornavirus proteins is based on their position in the viral RNA genome but does not necessarily imply a conserved function of proteins of different

  7. Interspecific variation of intracellular localization and postirradiation movement of Ku70-protein in fibroblastic cells

    International Nuclear Information System (INIS)

    Endoh, Daiji; Hayashi, Masanobu; Okui, Toyo; Kawase, Shiro; Kon, Yasushiro

    2003-01-01

    Ku (Ku70 and Ku80) Proteins are known as components of DNA-dependent protein kinase (DNA-PK) and play an important role for DNA repair. We previously reported that more than 70% of Ku proteins were located in cytoplasm of rat cells, the Ku proteins moved into nuclei of normal rat cells after X-irradiation, Ku proteins also moved into nuclei after X-irradiation but were not retained in nucleus of radiosensitive LEC rat cells. While reports have been shown about mechanisms on nuclear localization of Ku proteins, how Ku proteins export from nucleus is poorly understood. Here we show that C-terminal region of Ku70 protein is important for its cytoplasmic localization. When transfected into LEC rat cells, exogenous intact Ku70 (1-609) tagged with enhanced green fluorescent protein (EGFP-Ku70) localized mainly in the cytoplasm, whereas C-terminal-deletion mutant of Ku70 (1-593) tagged with EGFP (EGFP-Ku70D) was mainly localized in the nucleus. After X-irradiation, the endogenous intact EGFP-Ku70 once moved into nucleus, but returned into the cytoplasm. On the other hand, EGFP-Ku70D was retained in nucleus for two hours after X-irradiation. These results suggest that C-terminal region of Ku70 is included in the postirradiation nuclear export. Next, we investigated the intracellular localization of Ku70 proteins and the movement after X-irradiation of fibroblastic cells prepared from some mammalian species. Ku70 proteins were localized in nucleus and the postirradiation-extranuclear transport was not observed in human and African green monkey cells. On the other hand, Ku70 proteins were mainly localized in cytoplasm and moved into nucleus in mouse, Chinese hamster, Golden hamster, cotton rat, squirrel, cat and dog cells. These results may show that alternatively Ku70 protein is localized in the cytoplasm or nucleus depends on species and translocation of cytoplasmic Ku70 into nucleus is a response against low dose irradiation in fibroblasts of rodents, cats and dogs

  8. A mesoporous silica nanoparticle with charge-convertible pore walls for efficient intracellular protein delivery

    Energy Technology Data Exchange (ETDEWEB)

    Park, Hee Sung; Kwon, Ick Chan [Biomedical Research Center, Korea Institute of Science and Technology, Seoul 136-791 (Korea, Republic of); Kim, Chan Woo; Lee, Hong Jae; Yun, Young-Pil; Lee, Sang Cheon [Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Choi, Ji Hye [Department of Chemical and Biochemical Engineering, Yonsei University, Seoul 120-749 (Korea, Republic of); Lee, Se Geun [Department of Nano Technology, Advanced Nano Materials Research Team, Daegu Gyeongbuk Institute of Science and Technology, Daegu 704-230 (Korea, Republic of); Lee, Seung Jin [Department of Pharmacy, College of Pharmacy, Ewha Womans University, Seoul 120-750 (Korea, Republic of); Jeong, Seo Young, E-mail: syjeong@khu.ac.kr, E-mail: schlee@khu.ac.kr [Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 130-701 (Korea, Republic of)

    2010-06-04

    We report a smart mesoporous silica nanoparticle (MSN) with a pore surface designed to undergo charge conversion in intracellular endosomal condition. The surface of mesopores in the silica nanoparticles was engineered to have pH-hydrolyzable citraconic amide. Solid-state nuclear magnetic resonance (NMR), Fourier-transform infrared (FT-IR) spectroscopy, and Brunauer-Emmett-Teller (BET) analyses confirmed the successful modification of the pore surfaces. MSNs (MSN-Cit) with citraconic amide functionality on the pore surfaces exhibited a negative zeta potential (-10 mV) at pH 7.4 because of the presence of carboxylate end groups. At cellular endosomal pH ({approx}5.0), MSN-Cit have a positive zeta potential (16 mV) indicating the dramatic charge conversion from negative to positive by hydrolysis of surface citraconic amide. Cytochrome c (Cyt c) of positive charges could be incorporated into the pores of MSN-Cit by electrostatic interactions. The release of Cyt c can be controlled by adjusting the pH of the release media. At pH 7.4, the Cyt c release was retarded, whereas, at pH 5.0, MSN-Cit facilitated the release of Cyt c. The released Cyt c maintained the enzymatic activity of native Cyt c. Hemolytic activity of MSN-Cit over red blood cells (RBCs) was more pronounced at pH 5.0 than at pH 7.0, indicating the capability of intracellular endosomal escape of MSN carriers. Confocal laser scanning microscopy (CLSM) studies showed that MSN-Cit effectively released Cyt c in endosomal compartments after uptake by cancer cells. The MSN developed in this work may serve as efficient intracellular carriers of many cell-impermeable therapeutic proteins.

  9. Refrigerated storage of platelets initiates changes in platelet surface marker expression and localization of intracellular proteins.

    Science.gov (United States)

    Wood, Ben; Padula, Matthew P; Marks, Denese C; Johnson, Lacey

    2016-10-01

    Platelets (PLTs) are currently stored at room temperature (22°C), which limits their shelf life, primarily due to the risk of bacterial growth. Alternatives to room temperature storage include PLT refrigeration (2-6°C), which inhibits bacterial growth, thus potentially allowing an extension of shelf life. Additionally, refrigerated PLTs appear more hemostatically active than conventional PLTs, which may be beneficial in certain clinical situations. However, the mechanisms responsible for this hemostatic function are not well characterized. The aim of this study was to assess the protein profile of refrigerated PLTs in an effort to understand these functional consequences. Buffy coat PLTs were pooled, split, and stored either at room temperature (20-24°C) or under refrigerated (2-6°C) conditions (n = 8 in each group). PLTs were assessed for changes in external receptor expression and actin filamentation using flow cytometry. Intracellular proteomic changes were assessed using two-dimensional gel electrophoresis and Western blotting. PLT refrigeration significantly reduced the abundance of glycoproteins (GPIb, GPIX, GPIIb, and GPIV) on the external membrane. However, refrigeration resulted in the increased expression of high-affinity integrins (αIIbβ3 and β1) and activation and apoptosis markers (CD62P, CD63, and phosphatidylserine). PLT refrigeration substantially altered the abundance and localization of several cytoskeletal proteins and resulted in an increase in actin filamentation, as measured by phalloidin staining. Refrigerated storage of PLTs induces significant changes in the expression and localization of both surface-expressed and intracellular proteins. Understanding these proteomic changes may help to identify the mechanisms resulting in the refrigeration-associated alterations in PLT function and clearance. © 2016 AABB.

  10. Intracellular electric field and pH optimize protein localization and movement.

    Science.gov (United States)

    Cunningham, Jessica; Estrella, Veronica; Lloyd, Mark; Gillies, Robert; Frieden, B Roy; Gatenby, Robert

    2012-01-01

    Mammalian cell function requires timely and accurate transmission of information from the cell membrane (CM) to the nucleus (N). These pathways have been intensively investigated and many critical components and interactions have been identified. However, the physical forces that control movement of these proteins have received scant attention. Thus, transduction pathways are typically presented schematically with little regard to spatial constraints that might affect the underlying dynamics necessary for protein-protein interactions and molecular movement from the CM to the N. We propose messenger protein localization and movements are highly regulated and governed by Coulomb interactions between: 1. A recently discovered, radially directed E-field from the NM into the CM and 2. Net protein charge determined by its isoelectric point, phosphorylation state, and the cytosolic pH. These interactions, which are widely applied in elecrophoresis, provide a previously unknown mechanism for localization of messenger proteins within the cytoplasm as well as rapid shuttling between the CM and N. Here we show these dynamics optimize the speed, accuracy and efficiency of transduction pathways even allowing measurement of the location and timing of ligand binding at the CM--previously unknown components of intracellular information flow that are, nevertheless, likely necessary for detecting spatial gradients and temporal fluctuations in ligand concentrations within the environment. The model has been applied to the RAF-MEK-ERK pathway and scaffolding protein KSR1 using computer simulations and in-vitro experiments. The computer simulations predicted distinct distributions of phosphorylated and unphosphorylated components of this transduction pathway which were experimentally confirmed in normal breast epithelial cells (HMEC).

  11. Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

    Directory of Open Access Journals (Sweden)

    Unzueta U

    2012-08-01

    Full Text Available Ugutz Unzueta,1–3 María Virtudes Céspedes,3,4 Neus Ferrer-Miralles,1–3 Isolda Casanova,3,4 Juan Cedano,5 José Luis Corchero,1–3 Joan Domingo-Espín,1–3 Antonio Villaverde,1–3 Ramón Mangues,3,4 Esther Vázquez1–31Institut de Biotecnologia i de Biomedicina, 2Departamento de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, 3CIBER en Bioingeniería, Biomateriales y Nanomedicina, Bellaterra, Barcelona, 4Oncogenesis and Antitumor Drug Group, Biomedical Research Institute Sant Pau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 5Laboratory of Immunology, Regional Norte, Universidad de la Republica, Salto, UruguayBackground: Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4 is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing.Results: Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer.Conclusion: Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles

  12. Brucella Intracellular Life Relies on the Transmembrane Protein CD98 Heavy Chain.

    Science.gov (United States)

    Keriel, Anne; Botella, Eric; Estrach, Soline; Bragagnolo, Gabriel; Vergunst, Annette C; Feral, Chloe C; O'Callaghan, David

    2015-06-01

    Brucella are intracellular bacterial pathogens that use a type IV secretion system (T4SS) to escape host defenses and create a niche in which they can multiply. Although the importance of Brucella T4SS is clear, little is known about its interactions with host cell structures. In this study, we identified the eukaryotic protein CD98hc as a partner for Brucella T4SS subunit VirB2. This transmembrane glycoprotein is involved in amino acid transport, modulation of integrin signaling, and cell-to-cell fusion. Knockdown of CD98hc expression in HeLa cells demonstrated that it is essential for Brucella infection. Using knockout dermal fibroblasts, we confirmed its role for Brucella but found that it is not required for Salmonella infection. CD98hc transiently accumulates around the bacteria during the early phases of infection and is required for both optimal bacterial uptake and intracellular multiplication of Brucella. These results provide new insights into the complex interplay between Brucella and its host. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  13. Convergent regulation of skeletal muscle Ca2+ channels by dystrophin, the actin cytoskeleton, and cAMP-dependent protein kinase

    Science.gov (United States)

    Johnson, Barry D.; Scheuer, Todd; Catterall, William A.

    2005-03-01

    The skeletal muscle L-type Ca2+ channel (CaV1.1), which is responsible for initiating muscle contraction, is regulated by phosphorylation by cAMP-dependent protein kinase (PKA) in a voltage-dependent manner that requires direct physical association between the channel and the kinase mediated through A-kinase anchoring proteins (AKAPs). The role of the actin cytoskeleton in channel regulation was investigated in skeletal myocytes cultured from wild-type mice, mdx mice that lack the cytoskeletal linkage protein dystrophin, and a skeletal muscle cell line, 129 CB3. Voltage dependence of channel activation was shifted positively, and potentiation was greatly diminished in mdx myocytes and in 129 CB3 cells treated with the microfilament stabilizer phalloidin. Voltage-dependent potentiation by strong depolarizing prepulses was reduced in mdx myocytes but could be restored by positively shifting the stimulus potentials to compensate for the positive shift in the voltage dependence of gating. Inclusion of PKA in the pipette caused a negative shift in the voltage dependence of activation and restored voltage-dependent potentiation in mdx myocytes. These results show that skeletal muscle Ca2+ channel activity and voltage-dependent potentiation are controlled by PKA and microfilaments in a convergent manner. Regulation of Ca2+ channel activity by hormones and neurotransmitters that use the PKA signal transduction pathway may interact in a critical way with the cytoskeleton and may be impaired by deletion of dystrophin, contributing to abnormal regulation of intracellular calcium concentrations in dystrophic muscle.

  14. Rapamycin-binding FKBP25 associates with diverse proteins that form large intracellular entities

    Energy Technology Data Exchange (ETDEWEB)

    Galat, Andrzej, E-mail: galat@dsvidf.cea.fr; Thai, Robert

    2014-08-08

    Highlights: • The hFKBP25 interacts with diverse components of macromolecular entities. • We show that the endogenous human FKBP25 is bound to polyribosomes. • The endogenous hFKBP25 co-immunoprecipitated with nucleosomal proteins. • FKBP25 could induce conformational switch in macromolecular complexes. - Abstract: In this paper, we show some evidence that a member of the FK506-binding proteins, FKBP25 is associated to diverse components that are part of several different intracellular large-molecular mass entities. The FKBP25 is a high-affinity rapamycin-binding immunophilin, which has nuclear translocation signals present in its PPIase domain but it was detected both in the cytoplasm compartment and in the nuclear proteome. Analyses of antiFKBP25-immunoprecipitated proteins have revealed that the endogenous FKBP25 is associated to the core histones of the nucleosome, and with several proteins forming spliceosomal complexes and ribosomal subunits. Using polyclonal antiFKBP25 we have detected FKBP25 associated with polyribosomes. Added RNAs or 0.5 M NaCl release FKBP25 that was associated with the polyribosomes indicating that the immunophilin has an intrinsic capacity to form complexes with polyribonucleotides via its charged surface patches. Rapamycin or FK506 treatments of the polyribosomes isolated from porcine brain, HeLa and K568 cells caused a residual release of the endogenous FKBP25, which suggests that the immunophilin also binds to some proteins via its PPIase cavity. Our proteomics study indicates that the nuclear pool of the FKBP25 targets various nuclear proteins that are crucial for packaging of DNA, chromatin remodeling and pre-mRNA splicing whereas the cytosolic pool of this immunophilin is bound to some components of the ribosome.

  15. Rapamycin-binding FKBP25 associates with diverse proteins that form large intracellular entities

    International Nuclear Information System (INIS)

    Galat, Andrzej; Thai, Robert

    2014-01-01

    Highlights: • The hFKBP25 interacts with diverse components of macromolecular entities. • We show that the endogenous human FKBP25 is bound to polyribosomes. • The endogenous hFKBP25 co-immunoprecipitated with nucleosomal proteins. • FKBP25 could induce conformational switch in macromolecular complexes. - Abstract: In this paper, we show some evidence that a member of the FK506-binding proteins, FKBP25 is associated to diverse components that are part of several different intracellular large-molecular mass entities. The FKBP25 is a high-affinity rapamycin-binding immunophilin, which has nuclear translocation signals present in its PPIase domain but it was detected both in the cytoplasm compartment and in the nuclear proteome. Analyses of antiFKBP25-immunoprecipitated proteins have revealed that the endogenous FKBP25 is associated to the core histones of the nucleosome, and with several proteins forming spliceosomal complexes and ribosomal subunits. Using polyclonal antiFKBP25 we have detected FKBP25 associated with polyribosomes. Added RNAs or 0.5 M NaCl release FKBP25 that was associated with the polyribosomes indicating that the immunophilin has an intrinsic capacity to form complexes with polyribonucleotides via its charged surface patches. Rapamycin or FK506 treatments of the polyribosomes isolated from porcine brain, HeLa and K568 cells caused a residual release of the endogenous FKBP25, which suggests that the immunophilin also binds to some proteins via its PPIase cavity. Our proteomics study indicates that the nuclear pool of the FKBP25 targets various nuclear proteins that are crucial for packaging of DNA, chromatin remodeling and pre-mRNA splicing whereas the cytosolic pool of this immunophilin is bound to some components of the ribosome

  16. Inhibition of G protein-activated inwardly rectifying K+ channels by different classes of antidepressants.

    Directory of Open Access Journals (Sweden)

    Toru Kobayashi

    Full Text Available Various antidepressants are commonly used for the treatment of depression and several other neuropsychiatric disorders. In addition to their primary effects on serotonergic or noradrenergic neurotransmitter systems, antidepressants have been shown to interact with several receptors and ion channels. However, the molecular mechanisms that underlie the effects of antidepressants have not yet been sufficiently clarified. G protein-activated inwardly rectifying K(+ (GIRK, Kir3 channels play an important role in regulating neuronal excitability and heart rate, and GIRK channel modulation has been suggested to have therapeutic potential for several neuropsychiatric disorders and cardiac arrhythmias. In the present study, we investigated the effects of various classes of antidepressants on GIRK channels using the Xenopus oocyte expression assay. In oocytes injected with mRNA for GIRK1/GIRK2 or GIRK1/GIRK4 subunits, extracellular application of sertraline, duloxetine, and amoxapine effectively reduced GIRK currents, whereas nefazodone, venlafaxine, mianserin, and mirtazapine weakly inhibited GIRK currents even at toxic levels. The inhibitory effects were concentration-dependent, with various degrees of potency and effectiveness. Furthermore, the effects of sertraline were voltage-independent and time-independent during each voltage pulse, whereas the effects of duloxetine were voltage-dependent with weaker inhibition with negative membrane potentials and time-dependent with a gradual decrease in each voltage pulse. However, Kir2.1 channels were insensitive to all of the drugs. Moreover, the GIRK currents induced by ethanol were inhibited by sertraline but not by intracellularly applied sertraline. The present results suggest that GIRK channel inhibition may reveal a novel characteristic of the commonly used antidepressants, particularly sertraline, and contributes to some of the therapeutic effects and adverse effects.

  17. Crystal Structure of the Mammalian GIRK2 K+ Channel and Gating Regulation by G-Proteins, PIP2 and Sodium

    Science.gov (United States)

    Whorton, Matthew R.; MacKinnon, Roderick

    2011-01-01

    Summary G-protein-gated K+ channels (Kir3.1–Kir3.4) control electrical excitability in many different cells. Among their functions relevant to human physiology and disease, they regulate the heart rate and govern a wide range of neuronal activities. Here we present the first crystal structures of a G-protein-gated K+ channel. By comparing the wild-type structure to that of a constitutively active mutant, we identify a global conformational change through which G-proteins could open a G-loop gate in the cytoplasmic domain. The structures of both channels in the absence and presence of PIP2 show that G-proteins open only the G-loop gate in the absence of PIP2, but in the presence of PIP2 the G-loop gate and a second inner helix gate become coupled, so that both gates open. We also identify a strategically located Na+ ion-binding site, which would allow intracellular Na+ to modulate GIRK channel activity. These data provide a mechanistic description of multi-ligand regulation of GIRK channel gating. PMID:21962516

  18. Combining intracellular and secreted bioluminescent reporter proteins for multicolor cell-based assays.

    Science.gov (United States)

    Michelini, Elisa; Cevenini, Luca; Mezzanotte, Laura; Ablamsky, Danielle; Southworth, Tara; Branchini, Bruce R; Roda, Aldo

    2008-02-01

    Bioluminescent (BL) proteins are a promising tool for diverse applications based on reporter gene technology thanks to their high sensitivity and range of linear response. Due to their widespread use in the environmental, medical and agro-food fields, there is a great need for new BL reporter proteins with improved characteristics to provide researches a wide range of suitable reporters. Few efforts have been made in this direction and further improvement of BL reporter features (e.g., thermostability, narrower emission bandwidth, emission at different wavelengths) tailored for specific applications would be a remarkable progress toward the development of ultrasensitive multiplexed assays either in vitro or in vivo. The suitability of using red- and green-emitting thermostable mutants of Photinus pyralis firefly luciferase and two click beetle luciferases in combination with a secreted luciferase from Gaussia princeps was evaluated to develop a triple-color mammalian assay. Two triple-reporter model mammalian systems were developed in a human hepatoblastoma cell line to monitor the transcriptional regulation of cholesterol 7-alpha hydroxylase (cyp7a1), the enzyme that catalyzes the first and rate-limiting step of the main pathway responsible for cholesterol degradation in humans. These model systems allowed us to evaluate the feasibility of using two intracellular BL reporters and a secreted one in the same cell-based assay. The selection of reporter proteins characterized by similar expression levels was identified as a critical point for the development of a multicolor assay.

  19. FAM83H mutations cause ADHCAI and alter intracellular protein localization.

    Science.gov (United States)

    Lee, S-K; Lee, K-E; Jeong, T-S; Hwang, Y-H; Kim, S; Hu, J C-C; Simmer, J P; Kim, J-W

    2011-03-01

    Mutations in a family with sequence similarity 83 member H (FAM83H) cause autosomal-dominant hypocalcification amelogenesis imperfecta (ADH CAI). All FAM83H ADHCAI-causing mutations terminate translation or shift the reading frame within the specific exon 5 segment that encodes from Ser(287) to Glu(694). Mutations near Glu(694) cause a milder, more localized phenotype. We identified disease-causing FAM83H mutations in two families with ADHCAI: family 1 (g.3115C>T, c.1993 C>T, p.Q665X) and family 2 (g.3151C>T, c.2029 C>T, p.Q677X). We also tested the hypothesis that truncation mutations alter the intracellular localization of FAM83H. Wild-type FAM83H and p.E694X mutant FAM83H fused to green fluorescent protein (GFP) localized in the cytoplasm of HEK293T cells, but the mutant FAM83H proteins (p.R325X, p.W460X, and p.Q677X) fused to GFP localized mainly in the nucleus with slight expression in the cytoplasm. We conclude that nuclear targeting of the truncated FAM83H protein contributes to the severe, generalized enamel phenotype.

  20. 14-3-3 Proteins Buffer Intracellular Calcium Sensing Receptors to Constrain Signaling.

    Directory of Open Access Journals (Sweden)

    Michael P Grant

    Full Text Available Calcium sensing receptors (CaSR interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium.

  1. Medium pH in submerged cultivation modulates differences in the intracellular protein profile of Fusarium oxysporum.

    Science.gov (United States)

    da Rosa-Garzon, Nathália Gonsales; Laure, Hélen Julie; Souza-Motta, Cristina Maria de; Rosa, José César; Cabral, Hamilton

    2017-08-09

    Fusarium oxysporum is a filamentous fungus that damages a wide range of plants and thus causes severe crop losses. In fungal pathogens, the genes and proteins involved in virulence are known to be controlled by environmental pH. Here, we report the influence of culture-medium pH (5, 6, 7, and 8) on the production of degradative enzymes involved in the pathogenesis of F. oxysporum URM 7401 and on the 2D-electrophoresis profile of intracellular proteins in this fungus. F. oxysporum URM 7401 was grown in acidic, neutral, and alkaline culture media in a submerged bioprocess. After 96 hr, the crude extract was processed to enzyme activity assays, while the intracellular proteins were obtained from mycelium and analyzed using 2D electrophoresis and mass spectrometry. We note that the diversity of secreted enzymes was changed quantitatively in different culture-medium pH. Also, the highest accumulated biomass and the intracellular protein profile of F. oxysporum URM 7401 indicate an increase in metabolism in neutral-alkaline conditions. The differential profiles of secreted enzymes and intracellular proteins under the evaluated conditions indicate that the global protein content in F. oxysporum URM 7401 is modulated by extracellular pH.

  2. Electrophysiological Features of Single Store-Operated Calcium Channels in HEK S4 Cell Line with Stable STIM1 Protein Knockdown.

    Science.gov (United States)

    Shalygin, A V; Vigont, V A; Glushankova, L N; Zimina, O A; Kolesnikov, D O; Skopin, A Yu; Kaznacheeva, E V

    2017-07-01

    An important role in intracellular calcium signaling is played by store-operated channels activated by STIM proteins, calcium sensors of the endoplasmic reticulum. In stable STIM1 knockdown HEK S4 cells, single channels activated by depletion of intracellular calcium stores were detected by cell-attached patch-clamp technique and their electrophysiological parameters were described. Comparison of the properties of single channels in HEK293 and HEK S4 cells revealed no significant differences in their current-voltage curves, while regulation of store-operated calcium channels in these cell lines depended on the level of STIM1 expression. We can conclude that electrophysiological peculiarities of store-regulated calcium entry observed in different cells can be explained by differences in STIM1 expression.

  3. Accelerated microevolution in an outer membrane protein (OMP of the intracellular bacteria Wolbachia

    Directory of Open Access Journals (Sweden)

    Russell Jacob A

    2010-02-01

    Full Text Available Abstract Background Outer membrane proteins (OMPs of Gram-negative bacteria are key players in the biology of bacterial-host interactions. However, while considerable attention has been given to OMPs of vertebrate pathogens, relatively little is known about the role of these proteins in bacteria that primarily infect invertebrates. One such OMP is found in the intracellular bacteria Wolbachia, which are widespread symbionts of arthropods and filarial nematodes. Recent experimental studies have shown that the Wolbachia surface protein (WSP can trigger host immune responses and control cell death programming in humans, suggesting a key role of WSP for establishment and persistence of the symbiosis in arthropods. Results Here we performed an analysis of 515 unique alleles found in 831 Wolbachia isolates, to investigate WSP structure, microevolution and population genetics. WSP shows an eight-strand transmembrane β-barrel structure with four extracellular loops containing hypervariable regions (HVRs. A clustering approach based upon patterns of HVR haplotype diversity was used to group similar WSP sequences and to estimate the relative contribution of mutation and recombination during early stages of protein divergence. Results indicate that although point mutations generate most of the new protein haplotypes, recombination is a predominant force triggering diversity since the very first steps of protein evolution, causing at least 50% of the total amino acid variation observed in recently diverged proteins. Analysis of synonymous variants indicates that individual WSP protein types are subject to a very rapid turnover and that HVRs can accommodate a virtually unlimited repertoire of peptides. Overall distribution of WSP across hosts supports a non-random association of WSP with the host genus, although extensive horizontal transfer has occurred also in recent times. Conclusions In OMPs of vertebrate pathogens, large recombination impact, positive

  4. Channelopathies from mutations in the cardiac sodium channel protein complex.

    Science.gov (United States)

    Adsit, Graham S; Vaidyanathan, Ravi; Galler, Carla M; Kyle, John W; Makielski, Jonathan C

    2013-08-01

    The cardiac sodium current underlies excitability in heart, and inherited abnormalities of the proteins regulating and conducting this current cause inherited arrhythmia syndromes. This review focuses on inherited mutations in non-pore forming proteins of sodium channel complexes that cause cardiac arrhythmia, and the deduced mechanisms by which they affect function and dysfunction of the cardiac sodium current. Defining the structure and function of these complexes and how they are regulated will contribute to understanding the possible roles for this complex in normal and abnormal physiology and homeostasis. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes". Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Extraction of intracellular protein from Chlorella pyrenoidosa using a combination of ethanol soaking, enzyme digest, ultrasonication and homogenization techniques.

    Science.gov (United States)

    Zhang, Ruilin; Chen, Jian; Zhang, Xuewu

    2018-01-01

    Due to the rigid cell wall of Chlorella species, it is still challenging to effectively extract significant amounts of protein. Mass methods were used for the extraction of intracellular protein from microalgae with biological, mechanical and chemical approaches. In this study, based on comparison of different extraction methods, a new protocol was established to maximize extract amounts of protein, which was involved in ethanol soaking, enzyme digest, ultrasonication and homogenization techniques. Under the optimized conditions, 72.4% of protein was extracted from the microalgae Chlorella pyrenoidosa, which should contribute to the research and development of Chlorella protein in functional food and medicine. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. A Computational Approach to Studying Protein Folding Problems Considering the Crucial Role of the Intracellular Environment.

    Science.gov (United States)

    González-Pérez, Pedro P; Orta, Daniel J; Peña, Irving; Flores, Eduardo C; Ramírez, José U; Beltrán, Hiram I; Alas, Salomón J

    2017-10-01

    Intracellular protein folding (PF) is performed in a highly inhomogeneous, crowded, and correlated environment. Due to this inherent complexity, the study and understanding of PF phenomena is a fundamental issue in the field of computational systems biology. In particular, it is important to use a modeled medium that accurately reflects PF in natural systems. In the current study, we present a simulation wherein PF is carried out within an inhomogeneous modeled medium. Simulation resources included a two-dimensional hydrophobic-polar (HP) model, evolutionary algorithms, and the dual site-bond model. The dual site-bond model was used to develop an environment where HP beads could be folded. Our modeled medium included correlation lengths and fractal-like behavior, which were selected according to HP sequence lengths to induce folding in a crowded environment. Analysis of three benchmark HP sequences showed that the modeled inhomogeneous space played an important role in deeper energy folding and obtained better performance and convergence compared with homogeneous environments. Our computational approach also demonstrated that our correlated network provided a better space for PF. Thus, our approach represents a major advancement in PF simulations, not only for folding but also for understanding functional chemical structure and physicochemical properties of proteins in crowded molecular systems, which normally occur in nature.

  7. Evolution of the duplicated intracellular lipid-binding protein genes of teleost fishes.

    Science.gov (United States)

    Venkatachalam, Ananda B; Parmar, Manoj B; Wright, Jonathan M

    2017-08-01

    Increasing organismal complexity during the evolution of life has been attributed to the duplication of genes and entire genomes. More recently, theoretical models have been proposed that postulate the fate of duplicated genes, among them the duplication-degeneration-complementation (DDC) model. In the DDC model, the common fate of a duplicated gene is lost from the genome owing to nonfunctionalization. Duplicated genes are retained in the genome either by subfunctionalization, where the functions of the ancestral gene are sub-divided between the sister duplicate genes, or by neofunctionalization, where one of the duplicate genes acquires a new function. Both processes occur either by loss or gain of regulatory elements in the promoters of duplicated genes. Here, we review the genomic organization, evolution, and transcriptional regulation of the multigene family of intracellular lipid-binding protein (iLBP) genes from teleost fishes. Teleost fishes possess many copies of iLBP genes owing to a whole genome duplication (WGD) early in the teleost fish radiation. Moreover, the retention of duplicated iLBP genes is substantially higher than the retention of all other genes duplicated in the teleost genome. The fatty acid-binding protein genes, a subfamily of the iLBP multigene family in zebrafish, are differentially regulated by peroxisome proliferator-activated receptor (PPAR) isoforms, which may account for the retention of iLBP genes in the zebrafish genome by the process of subfunctionalization of cis-acting regulatory elements in iLBP gene promoters.

  8. Expression of Bax in yeast affects not only the mitochondria but also vacuolar integrity and intracellular protein traffic

    DEFF Research Database (Denmark)

    Dimitrova, Irina; Toby, Garabet G; Tili, Esmerina

    2004-01-01

    -transferase (BI-GST) leads to aggregation, but not fusion of the mitochondria. In addition, Bax affects the integrity of yeast vacuoles, resulting in the disintegration and eventual loss of the organelles, and the disruption of intracellular protein traffic. While Bcl-2 coexpression only partially corrects...

  9. Protein kinase CK2 is coassembled with small conductance ca(2+)-activated k(+) channels and regulates channel gating

    DEFF Research Database (Denmark)

    Bildl, Wolfgang; Strassmaier, Tim; Thurm, Henrike

    2004-01-01

    and serves as the Ca2+ sensor. Here we show that, in addition, the cytoplasmic N and C termini of the channel protein form a polyprotein complex with the catalytic and regulatory subunits of protein kinase CK2 and protein phosphatase 2A. Within this complex, CK2 phosphorylates calmodulin at threonine 80......, reducing by 5-fold the apparent Ca2+ sensitivity and accelerating channel deactivation. The results show that native SK channels are polyprotein complexes and demonstrate that the balance between kinase and phosphatase activities within the protein complex shapes the hyperpolarizing response mediated by SK...

  10. S100A4 and BMP-2 Co-Dependently Induce Vascular Smooth Muscle Cell Migration via pERK and Chloride Intracellular Channel 4 (CLIC4)

    Science.gov (United States)

    Spiekerkoetter, Edda; Guignabert, Christophe; de Jesus Perez, Vinicio; Alastalo, Tero-Pekka; Powers, Janine M; Wang, Lingli; Lawrie, Allan; Ambartsumian, Noona; Schmidt, Ann-Marie; Berryman, Mark; Ashley, Richard H; Rabinovitch, Marlene

    2009-01-01

    Rationale S100A4/Mts1 is implicated in motility of human pulmonary artery smooth muscle cells (hPASMC), through an interaction with the receptor for advanced glycation end products (RAGE). Objective We hypothesized that S100A4/Mts1-mediated hPASMC motility might be enhanced by loss of function of bone morphogenetic protein (BMP) receptor (R) II, observed in pulmonary arterial hypertension (PAH). Methods and Results Both S100A4/Mts1 (500ng/ml) and BMP-2 (10ng/ml) induce migration of hPASMCS in a novel co-dependent manner, in that the response to either ligand is lost with anti-RAGE or BMPRII siRNA. Phosphorylation of ERK is induced by both ligands and is required for motility by inducing MMP2 activity, but phosphoERK1/2 is blocked by anti-RAGE and not by BMPRII siRNA. In contrast, BMPRII siRNA, but not anti-RAGE, reduces expression of intracellular chloride channel 4 (CLIC4), a scaffolding molecule necessary for motility in response to S100A4/Mts1 or BMP-2. Reduced CLIC4 expression does not interfere with S100A4/Mts1 internalization or its interaction with myosin heavy chain IIA (MHCIIA), but does alter alignment of MHCIIA and actin filaments creating the appearance of vacuoles. This abnormality is associated with reduced peripheral distribution and/or delayed activation of RhoA and Rac1, small GTPases required for retraction and extension of lamellipodiae in motile cells. Conclusions Our studies demonstrate how a single ligand (BMP-2 or S100A4/Mts1) can recruit multiple cell surface receptors to relay signals that coordinate events culminating in a functional response, i.e., cell motility. We speculate that this carefully controlled process limits signals from multiple ligands, but could be subverted in disease. PMID:19713532

  11. Pannexin1 channel proteins in the zebrafish retina have shared and unique properties.

    Directory of Open Access Journals (Sweden)

    Sarah Kurtenbach

    Full Text Available In mammals, a single pannexin1 gene (Panx1 is widely expressed in the CNS including the inner and outer retinae, forming large-pore voltage-gated membrane channels, which are involved in calcium and ATP signaling. Previously, we discovered that zebrafish lack Panx1 expression in the inner retina, with drPanx1a exclusively expressed in horizontal cells of the outer retina. Here, we characterize a second drPanx1 protein, drPanx1b, generated by whole-genome duplications during teleost evolution. Homology searches strongly support the presence of pannexin sequences in cartilaginous fish and provide evidence that pannexins evolved when urochordata and chordata evolution split. Further, we confirm Panx1 ohnologs being solely present in teleosts. A hallmark of differential expression of drPanx1a and drPanx1b in various zebrafish brain areas is the non-overlapping protein localization of drPanx1a in the outer and drPanx1b in the inner fish retina. A functional comparison of the evolutionary distant fish and mouse Panx1s revealed both, preserved and unique properties. Preserved functions are the capability to form channels opening at resting potential, which are sensitive to known gap junction and hemichannel blockers, intracellular calcium, extracellular ATP and pH changes. However, drPanx1b is unique due to its highly complex glycosylation pattern and distinct electrophysiological gating kinetics. The existence of two Panx1 proteins in zebrafish displaying distinct tissue distribution, protein modification and electrophysiological properties, suggests that both proteins fulfill different functions in vivo.

  12. Modeling and measuring intracellular fluxes of secreted recombinant protein in Pichia pastoris with a novel 34S labeling procedure

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    Hann Stephan

    2011-06-01

    Full Text Available Abstract Background The budding yeast Pichia pastoris is widely used for protein production. To determine the best suitable strategy for strain improvement, especially for high secretion, quantitative data of intracellular fluxes of recombinant protein are very important. Especially the balance between intracellular protein formation, degradation and secretion defines the major bottleneck of the production system. Because these parameters are different for unlimited growth (shake flask and carbon-limited growth (bioreactor conditions, they should be determined under "production like" conditions. Thus labeling procedures must be compatible with minimal production media and the usage of bioreactors. The inorganic and non-radioactive 34S labeled sodium sulfate meets both demands. Results We used a novel labeling method with the stable sulfur isotope 34S, administered as sodium sulfate, which is performed during chemostat culivations. The intra- and extracellular sulfur 32 to 34 ratios of purified recombinant protein, the antibody fragment Fab3H6, are measured by HPLC-ICP-MS. The kinetic model described here is necessary to calculate the kinetic parameters from sulfur ratios of consecutive samples as well as for sensitivity analysis. From the total amount of protein produced intracellularly (143.1 μg g-1 h-1 protein per yeast dry mass and time about 58% are degraded within the cell, 35% are secreted to the exterior and 7% are inherited to the daughter cells. Conclusions A novel 34S labeling procedure that enables in vivo quantification of intracellular fluxes of recombinant protein under "production like" conditions is described. Subsequent sensitivity analysis of the fluxes by using MATLAB, indicate the most promising approaches for strain improvement towards increased secretion.

  13. Intracellular localization of the pseudorabies virus large tegument protein pUL36.

    Science.gov (United States)

    Möhl, Britta S; Böttcher, Sindy; Granzow, Harald; Kuhn, Jana; Klupp, Barbara G; Mettenleiter, Thomas C

    2009-10-01

    Homologs of the essential large tegument protein pUL36 of herpes simplex virus 1 are conserved throughout the Herpesviridae, complex with pUL37, and form part of the capsid-associated "inner" tegument. pUL36 is crucial for transport of the incoming capsid to and docking at the nuclear pore early after infection as well as for virion maturation in the cytoplasm. Its extreme C terminus is essential for pUL36 function interacting with pUL25 on nucleocapsids to start tegumentation (K. Coller, J. Lee, A. Ueda, and G. Smith, J. Virol. 81:11790-11797, 2007). However, controversy exists about the cellular compartment in which pUL36 is added to the nascent virus particle. We generated monospecific rabbit antisera against four different regions spanning most of pUL36 of the alphaherpesvirus pseudorabies virus (PrV). By immunofluorescence and immunoelectron microscopy, we then analyzed the intracellular location of pUL36 after transient expression and during PrV infection. While reactivities of all four sera were comparable, none of them showed specific intranuclear staining during PrV infection. In immunoelectron microscopy, neither of the sera stained primary enveloped virions in the perinuclear cleft, whereas extracellular mature virus particles were extensively labeled. However, transient expression of pUL36 alone resulted in partial localization to the nucleus, presumably mediated by nuclear localization signals (NLS) whose functionality was demonstrated by fusion of the putative NLS to green fluorescent protein (GFP) and GFP-tagged pUL25. Since PrV pUL36 can enter the nucleus when expressed in isolation, the NLS may be masked during infection. Thus, our studies show that during PrV infection pUL36 is not detectable in the nucleus or on primary enveloped virions, correlating with the notion that the tegument of mature virus particles, including pUL36, is acquired in the cytosol.

  14. Neurosteroids block the increase in intracellular calcium level induced by Alzheimer’s β-amyloid protein in long-term cultured rat hippocampal neurons

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    Midori Kato-Negishi

    2008-03-01

    Full Text Available Midori Kato-Negishi1, Masahiro Kawahara21Department of Developmental Morphology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi, Tokyo 183- 8526, Japan; 2Department of Analytical Chemistry, School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, 1714-1 Yoshino-cho, Nobeoka-shi, Miyazaki 882-8508, JapanAbstract: The neurotoxicity of β-amyloid protein (AβP is implicated in the etiology of Alzheimer’s disease. We previously have demonstrated that AβP forms Ca2+-permeable pores on neuronal membranes, causes a marked increase in intracellular calcium level, and leads to neuronal death. Here, we investigated in detail the features of AβP-induced changes in intracellular Ca2+ level in primary cultured rat hippocampal neurons using a multisite Ca2+- imaging system with fura-2 as a fluorescent probe. Only a small fraction of short-term cultured hippocampal neurons (ca 1 week in vitro exhibited changes in intracellular Ca2+ level after AβP exposure. However, AβP caused an acute increase in intracellular Ca2+ level in long-term cultured neurons (ca 1 month in vitro. The responses to AβP were highly heterogeneous, and immunohistochemical analysis using an antibody to AβP revealed that AβP is deposited on some but not all neurons. Considering that the disruption of Ca2+ homeostasis is the primary event in AβP neurotoxicity, substances that protect neurons from an AβP-induced intracellular Ca2+ level increase may be candidates as therapeutic drugs for Alzheimer’s disease. In line with the search for such protective substances, we found that the preadministration of neurosteroids including dehydroepiandrosterone, dehydroepiandrosterone sulfate, and pregnenolone significantly inhibits the increase in intracellular calcium level induced by AβP. Our results suggest the possible significance of neurosteroids, whose levels are reduced in the elderly, in preventing AβP neurotoxicity

  15. Study of the interaction of potassium ion channel protein with micelle by molecular dynamics simulation

    Science.gov (United States)

    Shantappa, Anil; Talukdar, Keka

    2018-04-01

    Ion channels are proteins forming pore inside the body of all living organisms. This potassium ion channel known as KcsA channel and it is found in the each cell and nervous system. Flow of various ions is regulated by the function of the ion channels. The nerve ion channel protein with protein data bank entry 1BL8, which is basically an ion channel protein in Streptomyces Lividans and which is taken up to form micelle-protein system and the system is analyzed by using molecular dynamics simulation. Firstly, ion channel pore is engineered by CHARMM potential and then Micelle-protein system is subjected to molecular dynamics simulation. For some specific micelle concentration, the protein unfolding is observed.

  16. Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel

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    Milos B. Rokic

    2018-04-01

    Full Text Available P2X2 receptors (P2X2R exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.

  17. Perturbation of intracellular acyl-CoA metabolism induces the unfolded protein response pathway and autophagy in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Feddersen, Søren

    2008-01-01

    autophagy mainly is a response to the stress of nutrient limitation. In the present study, we demonstrate that perturbation of fatty acid synthesis and transport either through inhibition of fatty acid synthase (FAS) or by depleting cells for the acyl-CoA binding protein, Acb1p, leads to induction of Hac1p....... This and the facts that Acb1p-depleted cells are hypersensitive to the immunosuppressive drug rapamycin and accumulate the transcription factor Msn2p in  the nucleus, indicate that perturbation of intracellular acyl-CoA metabolism leads to  a starvation response that upregulate autophagy, which involves both Ras......Eukaryotic cells have developed several strategies to respond and adapt to changes in their intracellular and extracellular environment. The unfolded protein response (UPR) pathway is activated following accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER), whereas...

  18. Tris-hydroxymethyl-aminomethane enhances capsaicin-induced intracellular Ca2+ influx through transient receptor potential V1 (TRPV1 channels

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    Satoshi Murakami

    2016-02-01

    Full Text Available Non-selective transient receptor potential vanilloid (TRPV cation channels are activated by various insults, including exposure to heat, acidity, and the compound capsaicin, resulting in sensations of pain in the skin, visceral organs, and oral cavity. Recently, TRPV1 activation was also demonstrated in response to basic pH elicited by ammonia and intracellular alkalization. Tris-hydroxymethyl aminomethane (THAM is widely used as an alkalizing agent; however, the effects of THAM on TRPV1 channels have not been defined. In this study, we characterized the effects of THAM-induced TRPV1 channel activation in baby hamster kidney cells expressing human TRPV1 (hTRPV1 and the Ca2+-sensitive fluorescent sensor GCaMP2 by real-time confocal microscopy. Notably, both capsaicin (1 μM and pH 6.5 buffer elicited steep increases in the intracellular Ca2+ concentration ([Ca2+]i, while treatment with THAM (pH 8.5 alone had no effect. However, treatment with THAM (pH 8.5 following capsaicin application elicited a profound, long-lasting increase in [Ca2+]i that was completely inhibited by the TRPV1 antagonist capsazepine. Taken together, these results suggest that hTRPV1 pre-activation is required to provoke enhanced, THAM-induced [Ca2+]i increases, which could be a mechanism underlying pain induced by basic pH.

  19. Dual regulation of G proteins and the G-protein-activated K+ channels by lithium.

    Science.gov (United States)

    Farhy Tselnicker, Isabella; Tsemakhovich, Vladimir; Rishal, Ida; Kahanovitch, Uri; Dessauer, Carmen W; Dascal, Nathan

    2014-04-01

    Lithium (Li(+)) is widely used to treat bipolar disorder (BPD). Cellular targets of Li(+), such as glycogen synthase kinase 3β (GSK3β) and G proteins, have long been implicated in BPD etiology; however, recent genetic studies link BPD to other proteins, particularly ion channels. Li(+) affects neuronal excitability, but the underlying mechanisms and the relevance to putative BPD targets are unknown. We discovered a dual regulation of G protein-gated K(+) (GIRK) channels by Li(+), and identified the underlying molecular mechanisms. In hippocampal neurons, therapeutic doses of Li(+) (1-2 mM) increased GIRK basal current (Ibasal) but attenuated neurotransmitter-evoked GIRK currents (Ievoked) mediated by Gi/o-coupled G-protein-coupled receptors (GPCRs). Molecular mechanisms of these regulations were studied with heterologously expressed GIRK1/2. In excised membrane patches, Li(+) increased Ibasal but reduced GPCR-induced GIRK currents. Both regulations were membrane-delimited and G protein-dependent, requiring both Gα and Gβγ subunits. Li(+) did not impair direct activation of GIRK channels by Gβγ, suggesting that inhibition of Ievoked results from an action of Li(+) on Gα, probably through inhibition of GTP-GDP exchange. In direct binding studies, Li(+) promoted GPCR-independent dissociation of Gαi(GDP) from Gβγ by a Mg(2+)-independent mechanism. This previously unknown Li(+) action on G proteins explains the second effect of Li(+), the enhancement of GIRK's Ibasal. The dual effect of Li(+) on GIRK may profoundly regulate the inhibitory effects of neurotransmitters acting via GIRK channels. Our findings link between Li(+), neuronal excitability, and both cellular and genetic targets of BPD: GPCRs, G proteins, and ion channels.

  20. Cross talk between β subunits, intracellular Ca2+ signaling, and SNAREs in the modulation of CaV 2.1 channel steady-state inactivation.

    Science.gov (United States)

    Serra, Selma Angèlica; Gené, Gemma G; Elorza-Vidal, Xabier; Fernández-Fernández, José M

    2018-01-01

    Modulation of Ca V 2.1 channel activity plays a key role in interneuronal communication and synaptic plasticity. SNAREs interact with a specific synprint site at the second intracellular loop (LII-III) of the Ca V 2.1 pore-forming α 1A subunit to optimize neurotransmitter release from presynaptic terminals by allowing secretory vesicles docking near the Ca 2+ entry pathway, and by modulating the voltage dependence of channel steady-state inactivation. Ca 2+ influx through Ca V 2.1 also promotes channel inactivation. This process seems to involve Ca 2+ -calmodulin interaction with two adjacent sites in the α 1A carboxyl tail (C-tail) (the IQ-like motif and the Calmodulin-Binding Domain (CBD) site), and contributes to long-term potentiation and spatial learning and memory. Besides, binding of regulatory β subunits to the α interaction domain (AID) at the first intracellular loop (LI-II) of α 1A determines the degree of channel inactivation by both voltage and Ca 2+ . Here, we explore the cross talk between β subunits, Ca 2+ , and syntaxin-1A-modulated Ca V 2.1 inactivation, highlighting the α 1A domains involved in such process. β 3 -containing Ca V 2.1 channels show syntaxin-1A-modulated but no Ca 2+ -dependent steady-state inactivation. Conversely, β 2a -containing Ca V 2.1 channels show Ca 2+ -dependent but not syntaxin-1A-modulated steady-state inactivation. A LI-II deletion confers Ca 2+ -dependent inactivation and prevents modulation by syntaxin-1A in β 3 -containing Ca V 2.1 channels. Mutation of the IQ-like motif, unlike CBD deletion, abolishes Ca 2+ -dependent inactivation and confers modulation by syntaxin-1A in β 2a -containing Ca V 2.1 channels. Altogether, these results suggest that LI-II structural modifications determine the regulation of Ca V 2.1 steady-state inactivation either by Ca 2+ or by SNAREs but not by both. © 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and

  1. Nephrotoxicity of Bence-Jones proteins: correlation with endocytosis by BHK cells and intracellular movement

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    Ana Lucia Nicastri

    2001-06-01

    Full Text Available The aim of this investigation was to evaluate the endocytosis of two Bence-Jones proteins by renal cells in order to elucidate the interference of their physical and chemical characteristics on nephrotoxicity. Bence-Jones proteins (AK and GL were purified and isolated from the urine of two patients with multiple myeloma. The isotype of both proteins was characterised as being human monoclonal lambda light chain. The AK protein presented mainly an Ip>7.0, a high content of galactose and a low amount of sialic acid molecules. On the other hand, the GL protein presented a single band with an Ip of 4.3, a higher level of sialic acid and a reduced amount of galactose, in comparison with the AK protein. Baby Hamster Kidney (BHK cells were maintained in culture in bottles at 37ºC, using DMEM culture media supplemented with 10% of calf serum with a pH of 7.4. Once the monolayer was observed to be confluent, the BHK cells were incubated with the two proteins, dissolved in a serum-free medium for 1, 5, 15, 30, 60 minutes and 24 hours. Control cells were established omitting the incubation with Bence-Jones proteins, but maintaining all of the other conditions. After, this the cells were washed, trypsinised, centrifuged and fixed in a solution of 4% paraformaldehyde and 0.5% glutaraldehyde on a 0.1 M, pH 7.4 phosphate buffer. Cells were processed for immunocytochemical reactions by using protein A coupled with colloidal gold and further silver enhancement. Semi-thin sections of the pellets were obtained and submitted to the cytochemical reactions. Detection of labelling was made by using light microscopy. It was observed that GL protein tended to be directed towards a perinuclear position, whereas the AK protein tended to suffer lysosomal deviation, suggesting that there is a direct contribution of physical and chemical characteristics on intracellular direction taken by Bence-Jones proteins.O objetivo deste trabalho foi avaliar a endocitose de duas prote

  2. The Potential Role of Cell Penetrating Peptides in the Intracellular Delivery of Proteins for Therapy of Erythroid Related Disorders

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    Lefkothea C. Papadopoulou

    2013-01-01

    Full Text Available The erythroid related disorders (ERDs represent a large group of hematological diseases, which in most cases are attributed either to the deficiency or malfunction of biosynthetic enzymes or oxygen transport proteins. Current treatments for these disorders include histo-compatible erythrocyte transfusions or allogeneic hematopoietic stem cell (HSC transplantation. Gene therapy delivered via suitable viral vectors or genetically modified HSCs have been under way. Protein Transduction Domain (PTD technology has allowed the production and intracellular delivery of recombinant therapeutic proteins, bearing Cell Penetrating Peptides (CPPs, into a variety of mammalian cells. Remarkable progress in the field of protein transduction leads to the development of novel protein therapeutics (CPP-mediated PTs for the treatment of monogenetic and/or metabolic disorders. The “concept” developed in this paper is the intracellular protein delivery made possible via the PTD technology as a novel therapeutic intervention for treatment of ERDs. This can be achieved via four stages including: (i the production of genetically engineered human CPP-mediated PT of interest, since the corresponding native protein either is missing or is mutated in the erythroid progenitor cell (ErPCs or mature erythrocytes of patients; (ii isolation of target cells from the peripheral blood of the selected patients; (iii ex vivo transduction of cells with the CPP-mediated PT of interest; and (iv re-administration of the successfully transduced cells back into the same patients.

  3. Effects of modulators of AMP-activated protein kinase on TASK-1/3 and intracellular Ca2+ concentration in rat carotid body glomus cells

    Science.gov (United States)

    Kim, Donghee; Kang1,2, Dawon; Martin, Elizabeth A.; Kim, Insook; Carroll, John L.

    2014-01-01

    Acute hypoxia depolarizes carotid body chemoreceptor (glomus) cells and elevates intracellular Ca2+ concentration ([Ca2+]i). Recent studies suggest that AMP-activated protein kinase (AMPK) mediates these effects of hypoxia by inhibiting the background K+ channels such as TASK. Here we studied the effects of modulators of AMPK on TASK activity in cell-attached patches. Activators of AMPK (1 mM AICAR and 0.1–0.5 mM A769662) did not inhibit TASK activity or cause depolarization during acute (10 min) or prolonged (2–3 hr) exposure. Hypoxia inhibited TASK activity by ~70% in cells pretreated with AICAR or A769662. Both AICAR and A769662 (15–40 min) failed to increase [Ca2+]i in glomus cells. Compound C (40 µM), an inhibitor of AMPK, showed no effect on hypoxia-induced inhibition of TASK. AICAR and A769662 phosphorylated AMPKα in PC12 cells, and Compound C blocked the phosphorylation. Our results suggest that AMPK does not affect TASK activity and is not involved in hypoxia-induced elevation of intracellular [Ca2+] in isolated rat carotid body glomus cells. PMID:24530802

  4. Functional characterization of the protein C A267T mutation: evidence for impaired secretion due to defective intracellular transport

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    Tjeldhorn Lena

    2010-09-01

    Full Text Available Abstract Background Activated protein C (PC is a serine protease that regulates blood coagulation by inactivating coagulation factors Va and VIIIa. PC deficiency is an autosomally inherited disorder associated with a high risk of recurrent venous thrombosis. The aim of the study was to explore the mechanisms responsible for severe PC deficiency in a patient with the protein C A267T mutation by in-vitro expression studies. Results Huh7 and CHO-K1 cells were transiently transfected with expression vectors containing wild-type (WT PC and mutated PC (A267T PC cDNAs. PC mRNA levels were assessed by qRT-PCR and the PC protein levels were measured by ELISA. The mRNA levels of WT PC and A267T PC were similar, while the intracellular protein level of A267T PC was moderately decreased compared to WT PC. The secretion of A267T PC into the medium was severely impaired. No differences in molecular weights were observed between WT and A267T PC before and after treatment with endo-β-N-acetylglucosaminidase. Proteasomal and lysosomal degradations were examined using lactacystin and bafilomycin, respectively, and revealed that A267T PC was slightly more susceptible for proteasomal degradation than WT PC. Intracellular co-localization analysis indicated that A267T PC was mainly located in the endoplasmic reticulum (ER, whereas WT PC was observed in both ER and Golgi. Conclusions In contrast to what has been reported for other PC mutants, intracellular degradation of A267T PC was not the main/dominant mechanism underlying the reduced intracellular and secretion levels of PC. Our results indicate that the A267T mutation most likely caused misfolding of PC, which might lead to increased retention of the mutated PC in ER.

  5. A ligand channel through the G protein coupled receptor opsin.

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    Peter W Hildebrand

    Full Text Available The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7, and B (between TM5 and 6, respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 A and is between 11.6 and 3.2 A wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal beta-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90 degrees elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all

  6. Identification of Chloride Intracellular Channel Protein 3 as a Novel Gene Affecting Human Bone Formation

    DEFF Research Database (Denmark)

    Brum, A M; Leije, M; J, Schreuders-Koedam

    2017-01-01

    is diminished and more adipocytes are seen in the bone marrow, suggesting a shift in MSC lineage commitment. Identification of specific factors that stimulate osteoblast differentiation from human MSCs may deliver therapeutic targets to treat osteoporosis. The aim of this study was to identify novel genes...... an in vivo human bone formation model in which hMSCs lentivirally transduced with the CLIC3 overexpression construct were loaded onto a scaffold (hydroxyapatite-tricalcium-phosphate), implanted under the skin of NOD-SCID mice, and analyzed for bone formation 8 weeks later. CLIC3 overexpression led to a 15...

  7. Cloning, intracellular localization, and expression of the mammalian selenocysteine-containing protein SELENOI (SelI) in tumor cell lines.

    Science.gov (United States)

    Varlamova, E G; Goltyaev, M V; Novoselov, V I; Fesenko, E E

    2017-09-01

    The intracellular localization of human selenoprotein SelI and the degree of expression of its gene in different human tumor cell lines were determined. It was found that the SelI protein is present in the nucleus, cytoplasm, and endoplasmic reticulum and is absent in the nucleolus. Since the oxidative stress caused by a sharp increase in the content of free radicals in the body is one of the causes of malignant transformation, the study of the role of the trace element selenium and selenocysteine-containing proteins as antioxidants in carcinogenesis is of great scientific interest.

  8. Autoradiographic and cytochemical studies on the intracellular transport of secreted proteins in the lacrimal ducts (glandula extraorbitalis) of the rat

    International Nuclear Information System (INIS)

    Vogel, H.

    1982-01-01

    Azini was isolated from the glandula lacrimalis of the rat. Its vitality was proven by oxygen use measurements. In autoradiographic studies isolated Azini was marked with L-(4,5- 3 H)-leucine and fixed at various times thereafter. The light microscopic autoradiography showed a time dependent distribution of the silver grains whose association with membrane-enclosed compartments made the electron microscopic autoradiography possible. This distribution allows an analysis of the kinetics of the intracellular transport of secreted proteins. Because of its limited spatial resolution the autoradiographic research methods were combined with the cytochemical presentation of the peroxidase, a secreted protein, of the lacrimal duct. (orig./MG) [de

  9. Monitoring the intracellular pH of Zygosaccharomyces bailii by green fluorescent protein.

    Science.gov (United States)

    Dang, T D T; De Maeseneire, S L; Zhang, B Y; De Vos, W H; Rajkovic, A; Vermeulen, A; Van Impe, J F; Devlieghere, F

    2012-06-01

    It is generally known that intracellular pH (pH(i)) plays a vital role in cell physiology and that pH(i) homeostasis is essential for normal cellular functions. Therefore, it is desirable to know the pH(i) during cell life cycle or under various growth conditions. Different methods to measure pH(i) have been developed and among these methods, the use of pH-sensitive green fluorescent protein (GFP) as a pH(i) indicator is a promising technique. By using this approach, not only can more accurate pH(i) results be obtained but also long-term experiments on pH(i) can be performed. In this study, the wild type Zygosaccharomyces bailii, a notorious food spoilage yeast, was transformed with a plasmid encoding a pH-sensitive GFP (i.e. pHluorin), enabling the pH(i) of the yeast to be determined based on cellular fluorescent signals. After the transformation, growth and pH(i) of the yeast were investigated in four different acidic conditions at 22°C during 26days. From the experimental results, the transformation effectiveness was verified and a good correlation between yeast growth and pH(i) was noticed. Particularly, it was observed that the yeast has an ability to tolerate a significant pH(i) drop during exponential phase and a subsequent pH(i) recovery in stationary phase, which may underlie the exceptional acid resistance of the yeast. This was the first time that a GFP-based approach for pH(i) measurement was applied in Z. bailii and that the pH(i) of the yeast was monitored during such a long period (26days). It can be expected that greater understanding of the physiological properties and mechanisms behind the special acid resistance of the yeast will be obtained from further studies on this new yeast strain. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Both antiviral activity and intracellular localization of chicken Mx protein depend on a polymorphism at amino acid position 631.

    Science.gov (United States)

    Sasaki, Keisuke; Yoneda, Akihiro; Ninomiya, Akinori; Kawahara, Manabu; Watanabe, Tomomasa

    2013-01-04

    The Mx protein is known to inhibit the multiplication of several RNA viruses. In chickens, a polymorphism at amino acid position 631 (631 aa) of Mx protein has been suggested to be involved in the antiviral ability against vesicular stomatitis virus (VSV) and influenza virus, indicating that a Ser-to-Asn substitution at 631 aa is the source of this antiviral ability. However, how the substitution at 631 aa contributes to the antiviral activity remains to be clarified. In this study, we investigated differences in antiviral activity against VSV and intracellular localization between Ser and Asn types at 631 aa of the chicken Mx protein. The results showed that chicken Mx protein with an Asn at 631 aa inhibited VSV multiplication and Mx distribution in a granular-like pattern in the cytoplasm. However, Mx carrying the Ser type did not inhibit viral growth and homogenous spread throughout the cytoplasm. Furthermore, we found that replacing Ser with Asn at 631 aa provided Mx with antiviral activity against VSV, with Mx showing granular-like distribution in the cytoplasm. These results demonstrated that a single amino acid polymorphism at 631 aa of the chicken Mx protein altered both the antiviral activity and intracellular localization. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Plasma membrane-located purine nucleotide transport proteins are key components for host exploitation by microsporidian intracellular parasites.

    Directory of Open Access Journals (Sweden)

    Eva Heinz

    2014-12-01

    Full Text Available Microsporidia are obligate intracellular parasites of most animal groups including humans, but despite their significant economic and medical importance there are major gaps in our understanding of how they exploit infected host cells. We have investigated the evolution, cellular locations and substrate specificities of a family of nucleotide transport (NTT proteins from Trachipleistophora hominis, a microsporidian isolated from an HIV/AIDS patient. Transport proteins are critical to microsporidian success because they compensate for the dramatic loss of metabolic pathways that is a hallmark of the group. Our data demonstrate that the use of plasma membrane-located nucleotide transport proteins (NTT is a key strategy adopted by microsporidians to exploit host cells. Acquisition of an ancestral transporter gene at the base of the microsporidian radiation was followed by lineage-specific events of gene duplication, which in the case of T. hominis has generated four paralogous NTT transporters. All four T. hominis NTT proteins are located predominantly to the plasma membrane of replicating intracellular cells where they can mediate transport at the host-parasite interface. In contrast to published data for Encephalitozoon cuniculi, we found no evidence for the location for any of the T. hominis NTT transporters to its minimal mitochondria (mitosomes, consistent with lineage-specific differences in transporter and mitosome evolution. All of the T. hominis NTTs transported radiolabelled purine nucleotides (ATP, ADP, GTP and GDP when expressed in Escherichia coli, but did not transport radiolabelled pyrimidine nucleotides. Genome analysis suggests that imported purine nucleotides could be used by T. hominis to make all of the critical purine-based building-blocks for DNA and RNA biosynthesis during parasite intracellular replication, as well as providing essential energy for parasite cellular metabolism and protein synthesis.

  12. Intracellular and Extracellular Expression of Bacillus thuringiensis Crystal Protein Cry5B in Lactococcus lactis for Use as an Anthelminthic.

    Science.gov (United States)

    Durmaz, Evelyn; Hu, Yan; Aroian, Raffi V; Klaenhammer, Todd R

    2016-02-15

    The Bacillus thuringiensis crystal (Cry) protein Cry5B (140 kDa) and a truncated version of the protein, tCry5B (79 kDa), are lethal to nematodes. Genes encoding the two proteins were separately cloned into a high-copy-number vector with a strong constitutive promoter (pTRK593) in Lactococcus lactis for potential oral delivery against parasitic nematode infections. Western blots using a Cry5B-specific antibody revealed that constitutively expressed Cry5B and tCry5B were present in both cells and supernatants. To increase production, cry5B was cloned into the high-copy-number plasmid pMSP3535H3, carrying a nisin-inducible promoter. Immunoblotting revealed that 3 h after nisin induction, intracellular Cry5B was strongly induced at 200 ng/ml nisin, without adversely affecting cell viability or cell membrane integrity. Both Cry5B genes were also cloned into plasmid pTRK1061, carrying a promoter and encoding a transcriptional activator that invoke low-level expression of prophage holin and lysin genes in Lactococcus lysogens, resulting in a leaky phenotype. Cry5B and tCry5B were actively expressed in the lysogenic strain L. lactis KP1 and released into cell supernatants without affecting culture growth. Lactate dehydrogenase (LDH) assays indicated that Cry5B, but not LDH, leaked from the bacteria. Lastly, using intracellular lysates from L. lactis cultures expressing both Cry5B and tCry5B, in vivo challenges of Caenorhabditis elegans worms demonstrated that the Cry proteins were biologically active. Taken together, these results indicate that active Cry5B proteins can be expressed intracellularly in and released extracellularly from L. lactis, showing potential for future use as an anthelminthic that could be delivered orally in a food-grade microbe. Copyright © 2016 Durmaz et al.

  13. Intracellular and Extracellular Expression of Bacillus thuringiensis Crystal Protein Cry5B in Lactococcus lactis for Use as an Anthelminthic

    Science.gov (United States)

    Durmaz, Evelyn; Hu, Yan; Aroian, Raffi V.

    2015-01-01

    The Bacillus thuringiensis crystal (Cry) protein Cry5B (140 kDa) and a truncated version of the protein, tCry5B (79 kDa), are lethal to nematodes. Genes encoding the two proteins were separately cloned into a high-copy-number vector with a strong constitutive promoter (pTRK593) in Lactococcus lactis for potential oral delivery against parasitic nematode infections. Western blots using a Cry5B-specific antibody revealed that constitutively expressed Cry5B and tCry5B were present in both cells and supernatants. To increase production, cry5B was cloned into the high-copy-number plasmid pMSP3535H3, carrying a nisin-inducible promoter. Immunoblotting revealed that 3 h after nisin induction, intracellular Cry5B was strongly induced at 200 ng/ml nisin, without adversely affecting cell viability or cell membrane integrity. Both Cry5B genes were also cloned into plasmid pTRK1061, carrying a promoter and encoding a transcriptional activator that invoke low-level expression of prophage holin and lysin genes in Lactococcus lysogens, resulting in a leaky phenotype. Cry5B and tCry5B were actively expressed in the lysogenic strain L. lactis KP1 and released into cell supernatants without affecting culture growth. Lactate dehydrogenase (LDH) assays indicated that Cry5B, but not LDH, leaked from the bacteria. Lastly, using intracellular lysates from L. lactis cultures expressing both Cry5B and tCry5B, in vivo challenges of Caenorhabditis elegans worms demonstrated that the Cry proteins were biologically active. Taken together, these results indicate that active Cry5B proteins can be expressed intracellularly in and released extracellularly from L. lactis, showing potential for future use as an anthelminthic that could be delivered orally in a food-grade microbe. PMID:26682852

  14. Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration.

    Directory of Open Access Journals (Sweden)

    Patrick M Schaefer

    Full Text Available One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ, which derives from the processing of the amyloid precursor protein (APP. Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1, increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases.

  15. Evaluation of intracellular anion superoxide level, heat shock protein A2 and protamine positive spermatozoa percentages in teratoasthenozoospermia

    Directory of Open Access Journals (Sweden)

    Parvin Sabeti

    2017-09-01

    Full Text Available Background: Teratoasthenozoospermia (TA is a severe form of male infertility with no clear etiology. Objective: To compare the level of intracellular anion superoxide (O2–, heat shock protein A2 (HSPA2 and protamine deficiencies in ejaculated spermatozoa between teratoasthenozoospermic and normozoospermic men. Materials and Methods: In this case- control study, semen samples of 20 infertile men, with TA (with normal morphology lower than 4%_ and total motility lower than 40% as the case group and 20 normozoospermic fertile men as the control group were evaluated for intracellular O2 – and HSPA2 by flow cytometry and protamine deficiency by Chromomycin A3 (CMA3 test. Results: The rate of CMA3+ spermatozoa in the case group was higher than controls (p=0.001. The percentages of HSPA2+ spermatozoa in the cases were significantly lower than controls (p=0.001. Also, intracellular O2 – levels in the case group were significantly higher than controls (p=0.001 and had positive correlations with sperm apoptosis (r=0.79, p=0.01 and CMA3 positive sperm (r=0.76, p=0.01, but negative correlations with normal morphology (r=-0.81, p=0.01 and motility (r=-0.81, p=0.01. There was no significant correlation between intracellular O2 – and HSPA2 in the case group (r=0.041, p=0.79. Conclusion: We suggest that the increase in intracellular O2 –, decrease in spermatozoa HSPA2+, and high percentages of spermatozoa with immature chromatin might be considered as etiologies of infertility in TA patients

  16. Biosynthesis of intestinal microvillar proteins. Low temperature arrests both processing and intracellular transport

    DEFF Research Database (Denmark)

    Danielsen, E M; Hansen, Gert Helge; Cowell, G M

    1989-01-01

    The effect of culture at 20 degrees C on biosynthesis of microvillar enzymes was studied in pig small intestinal mucosal explants. At this temperature, aminopeptidase N (EC 3.4.11.2) and sucrase-isomaltase (EC 3.2.1.48-10) both accumulated intracellularly, predominantly in their transient, high m...

  17. The intracellular domain of the Drosophila cholinesterase-like neural adhesion protein, gliotactin, is natively unfolded

    NARCIS (Netherlands)

    Zeev-Ben-Mordehai, T; Rydberg, EH; Solomon, A.; Toker, L; Auld, VJ; Silman, I.; Botti, S; Sussman, J.L.

    2003-01-01

    Drosophila gliotactin (Gli) is a 109-kDa transmembrane, cholinesterase-like adhesion molecule (CLAM), expressed in peripheral glia, that is crucial for formation of the blood-nerve barrier. The intracellular portion (Gli-cyt) was cloned and expressed in the cytosolic fraction of Escherichia coli

  18. Biosynthesis of intestinal microvillar proteins. Low temperature arrests both processing and intracellular transport

    DEFF Research Database (Denmark)

    Danielsen, E M; Hansen, Gert Helge; Cowell, G M

    1989-01-01

    The effect of culture at 20 degrees C on biosynthesis of microvillar enzymes was studied in pig small intestinal mucosal explants. At this temperature, aminopeptidase N (EC 3.4.11.2) and sucrase-isomaltase (EC 3.2.1.48-10) both accumulated intracellularly, predominantly in their transient, high...

  19. Influences of dietary protein sources and crude protein levels on intracellular free amino acid profile in the longissimus dorsi muscle of finishing gilts.

    Science.gov (United States)

    Qin, Chunfu; Huang, Ping; Qiu, Kai; Sun, Wenjuan; Xu, Ling; Zhang, Xin; Yin, Jingdong

    2015-01-01

    The current study was carried out to determine effects of dietary protein source and crude protein (CP) level on carcass characteristics, meat quality, and muscle amino acid (AA) profile in finishing gilts. The experiment was designed as a 2 × 2 factorial arrangement with two sources of dietary proteins (cottonseed meal, CSM vs. soybean meal, SBM) and two levels of CP (12 % vs. 14 %, as-fed basis). Seventy-two crossbred gilts (89.5 ± 0.9 kg) were allotted to one of four dietary treatments in a randomized complete block design for a period of 28 d. All diets were formulated to be isoenergetic and similar concentrations of standardized ileal digestible essential AA covering the nutrient requirements of pigs. Growth, carcass characteristics and meat quality were not affected by dietary protein source nor crude protein level (P > 0.10) except that average daily feed intake was increased by CSM diets (P = 0.03). Gilts offered reduced protein diets had lower muscle pH45min (P gilts offered CSM diets, while muscle intracellular free valine concentration was increased (P = 0.03). The gilts offered reduced protein diets had greater intracellular concentrations of free methionine, lysine, and total AA in muscle (P < 0.05). These results suggest that CSM could replace SBM as a primary protein source in finishing pig diets in terms of performance, N efficiency, carcass characteristics, and meat quality, but decrease the concentrations of muscle specific AA. Furthermore, the reduced protein diet played an important role in increasing muscle intracellular concentrations of specific free amino acids (FAA), and in reducing the relative ratios of specific FAA to lysine in longissimus dorsi muscle of pig, whose biological meaning needs further studies.

  20. Structural analysis of the intracellular domain of (pro)renin receptor fused to maltose-binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Gao, Xiaoli [Department of Biochemistry and Molecular Biology, Michigan State University (United States); Michael Garavito, R., E-mail: garavito@msu.edu [Department of Biochemistry and Molecular Biology, Michigan State University (United States)

    2011-04-22

    Highlights: {yields} Crystal structure of the intracellular domain of (pro)renin receptor (PRR-IC) as MBP fusion protein at 2.0 A (maltose-free) and 2.15 A (maltose-bound). {yields} MBP fusion protein is a dimer in crystals in the presence and absence of maltose. {yields} PRR-IC domain is responsible for the dimerization of the fusion protein. {yields} Residues in the PRR-IC domain, particularly two tyrosines, dominate the intermolecular interactions, suggesting a role for the PRR-IC domain in PRR dimerization. -- Abstract: The (pro)renin receptor (PRR) is an important component of the renin-angiotensin system (RAS), which regulates blood pressure and cardiovascular function. The integral membrane protein PRR contains a large extracellular domain ({approx}310 amino acids), a single transmembrane domain ({approx}20 amino acids) and an intracellular domain ({approx}19 amino acids). Although short, the intracellular (IC) domain of the PRR has functionally important roles in a number of signal transduction pathways activated by (pro)renin binding. Meanwhile, together with the transmembrane domain and a small portion of the extracellular domain ({approx}30 amino acids), the IC domain is also involved in assembly of V{sub 0} portion of the vacuolar proton-translocating ATPase (V-ATPase). To better understand structural and multifunctional roles of the PRR-IC, we report the crystal structure of the PRR-IC domain as maltose-binding protein (MBP) fusion proteins at 2.0 A (maltose-free) and 2.15 A (maltose-bound). In the two separate crystal forms having significantly different unit-cell dimensions and molecular packing, MBP-PRR-IC fusion protein was found to be a dimer, which is different with the natural monomer of native MBP. The PRR-IC domain appears as a relatively flexible loop and is responsible for the dimerization of MBP fusion protein. Residues in the PRR-IC domain, particularly two tyrosines, dominate the intermonomer interactions, suggesting a role for the PRR

  1. Heat shock protein 90 has roles in intracellular calcium homeostasis, protein tyrosine phosphorylation regulation, and progesterone-responsive sperm function in human sperm.

    Science.gov (United States)

    Li, Kun; Xue, Yamei; Chen, Aijun; Jiang, Youfang; Xie, Haifeng; Shi, Qixian; Zhang, Songying; Ni, Ya

    2014-01-01

    Heat shock protein 90 plays critical roles in client protein maturation, signal transduction, protein folding and degradation, and morphological evolution; however, its function in human sperm is not fully understood. Therefore, our objective in this study was to elucidate the mechanism by which heat shock protein 90 exerts its effects on human sperm function. By performing indirect immunofluorescence staining, we found that heat shock protein 90 was localized primarily in the neck, midpiece, and tail regions of human sperm, and that its expression increased with increasing incubation time under capacitation conditions. Geldanamycin, a specific inhibitor of heat shock protein 90, was shown to inhibit this increase in heat shock protein 90 expression in western blotting analyses. Using a multifunctional microplate reader to examine Fluo-3 AM-loaded sperm, we observed for the first time that inhibition of heat shock protein 90 by using geldanamycin significantly decreased intracellular calcium concentrations during capacitation. Moreover, western blot analysis showed that geldanamycin enhanced tyrosine phosphorylation of several proteins, including heat shock protein 90, in a dose-dependent manner. The effects of geldanamycin on human sperm function in the absence or presence of progesterone was evaluated by performing chlortetracycline staining and by using a computer-assisted sperm analyzer. We found that geldanamycin alone did not affect sperm capacitation, hyperactivation, and motility, but did so in the presence of progesterone. Taken together, these data suggest that heat shock protein 90, which increases in expression in human sperm during capacitation, has roles in intracellular calcium homeostasis, protein tyrosine phosphorylation regulation, and progesterone-stimulated sperm function. In this study, we provide new insights into the roles of heat shock protein 90 in sperm function.

  2. Regulation of ATP-sensitive K+ channels in insulinoma cells: Activation by somatostatin and protein kinase C and the role of cAMP

    International Nuclear Information System (INIS)

    De Weille, J.R.; Schmid-Antomarchi, H.; Fosset, M.; Lazdunski, M.

    1989-01-01

    The actions of somatostatin and of the phorbol ester 4β-phorbol 12-myristate 13-acetate (PMA) were studied in rat insulinoma (RINm5F) cells by electrophysiological and 86 Rb + flux techniques. Both PMA and somatostatin hyperpolarize insulinoma cells by activating ATP-sensitive K + channels. The presence of intracellular GTP is required for the somatostatin effects. PMA- and somatostatin-induced hyperpolarization and channel activity are inhibited by the sulfonylurea glibenclamide. Glibenclamide-sensitive 86 Rb + efflux from insulinoma cells is stimulated by somatostatin in a dose-dependent manner (half maximal effect at 0.7 nM) and abolished by pertussis toxin pretreatment. Mutual roles of a GTP-binding protein, of protein kinase C, and of cAMP in the regulation of ATP-sensitive K + channels are discussed

  3. Biosynthesis of intestinal microvillar proteins. Intracellular processing of lactase-phlorizin hydrolase

    DEFF Research Database (Denmark)

    Danielsen, E M; Skovbjerg, H; Norén, Ove

    1984-01-01

    The biosynthesis of pig small intestinal lactase-phlorizin hydrolase (EC 3.2.1.23-62) was studied by labelling of organ cultured mucosal explants with [35S]methionine. The earliest detactable form of the enzyme was an intracellular, membrane-bound polypeptide of Mr 225 000, sensitive to endo H...... 000 polypeptide is of the same size as the mature lactase-phlorizin hydrolase and was the only form expressed in the microvillar membrane. Together, these data are indicative of an intracellular proteolytic cleavage during transport. The presence of leupeptin during labelling prevented the appearance...... of the Mr 160 000 form but not that of the Mr 245 000 polypeptide, suggesting that the proteolytic cleavage takes place after trimming and complex glycosylation. The proteolytic cleavage was not essential for the transport since the precursor was expressed in the microvillar membrane in the presence...

  4. Analysis of Immunogenicity of Intracellular CTAR Fragments of Epstein-Barr Virus Latent Phase Protein LMP1.

    Science.gov (United States)

    Lomakin, Ya A; Shmidt, A A; Bobik, T V; Chernov, A S; Pyrkov, A Yu; Aleksandrova, N M; Okunola, D O; Vaskina, M I; Ponomarenko, N A; Telegin, G B; Dubina, M V; Belogurov, A A

    2017-10-01

    Intracellular fragments of latent phase protein LMP1 of Epstein-Barr virus, denoted as CTAR1/2/3, can trigger a variety of cell cascades and contribute to the transforming potential of the virus. Generation of recombinant proteins CTAR1/2/3 is expected to yield more ample data on functional and immunogenic characteristics of LMP1. We created genetic constructs for prokaryotic expression of LMP1 CTAR fragments and selected optimal conditions for their production and purification. Using a new library of LMP1 CTAR fragments, we carried out epitope mapping of a diagnostic anti-LMP1 antibody S12. Analysis of polyclonal serum antibodies from mice immunized with full-length LMP1 confirmed immunogenicity of CTAR elements comparable with that of full-length protein.

  5. A novel role of the L-type calcium channel α1D subunit as a gatekeeper for intracellular zinc signaling: zinc wave.

    Directory of Open Access Journals (Sweden)

    Satoru Yamasaki

    Full Text Available Recent studies have shown that zinc ion (Zn can behave as an intracellular signaling molecule. We previously demonstrated that mast cells stimulated through the high-affinity IgE receptor (FcεRI rapidly release intracellular Zn from the endoplasmic reticulum (ER, and we named this phenomenon the "Zn wave". However, the molecules responsible for releasing Zn and the roles of the Zn wave were elusive. Here we identified the pore-forming α(1 subunit of the Cav1.3 (α(1D L-type calcium channel (LTCC as the gatekeeper for the Zn wave. LTCC antagonists inhibited the Zn wave, and an agonist was sufficient to induce it. Notably, α(1D was mainly localized to the ER rather than the plasma membrane in mast cells, and the Zn wave was impaired by α(1D knockdown. We further found that the LTCC-mediated Zn wave positively controlled cytokine gene induction by enhancing the DNA-binding activity of NF-κB. Consistent with this finding, LTCC antagonists inhibited the cytokine-mediated delayed-type allergic reaction in mice without affecting the immediate-type allergic reaction. These findings indicated that the LTCC α(1D subunit located on the ER membrane has a novel function as a gatekeeper for the Zn wave, which is involved in regulating NF-κB signaling and the delayed-type allergic reaction.

  6. Regulation of B cell differentiation by intracellular membrane associated proteins and microRNAs: role in the antibody response

    Directory of Open Access Journals (Sweden)

    Zheng eLou

    2015-10-01

    Full Text Available B cells are central to adaptive immunity and their functions in antibody responses are exquisitely regulated. As suggested by recent findings, B cell differentiation is mediated by intracellular membrane structures (including endosomes, lysosomes and autophagosomes and protein factors specifically associated with these membranes, including Rab7, Atg5 and Atg7. These factors participate in vesicle formation/trafficking, signal transduction and induction of gene expression to promote antigen presentation, CSR/SHM, and generation/maintenance of plasma cells and memory B cells. Their expression is induced in B cells activated to differentiate and further fine-tuned by immune-modulating microRNAs, which coordinates CSR/SHM, plasma cell differentiation and memory B cell differentiation. These short non-coding RNAs would individually target multiple factors associated with the same intracellular membrane compartments and collaboratively target a single factor in addition to regulate AID and Blimp-1. These, together with regulation of microRNA biogenesis and activities by endosomes and autophagosomes, show that intracellular membranes and microRNAs, two broadly relevant cell constituents, play important roles in balancing gene expression to specify B cell differentiation processes for optimal antibody responses.

  7. Construction of a novel glucose-sensing molecule based on a substrate-binding protein for intracellular sensing.

    Science.gov (United States)

    Sakaguchi-Mikami, Akane; Taniguchi, Akiyoshi; Sode, Koji; Yamazaki, Tomohiko

    2011-04-01

    A novel transcriptional regulator responding to glucose was designed with a substrate-binding protein (SBP) as a probe towards intracellular sensing system for glucose in mammalian cells. A chimeric protein of an SBP for glucose (GBP) and a LacI-type regulator, LacI (SLCP(GL) ), was designed, constructed and characterized using Escherichia coli recombinant protein. We report that SLCP(GL) has a glucose-specific binding ability and an operator-sequence specific DNA-binding ability. The loss of its DNA-binding ability in the presence of glucose suggests a role as a transcriptional regulator in vitro. The glucose-dependent gene regulation function of SLCP(GL) in cells was investigated using mammalian cells co-transfected with SLCP(GL) and Lac operator-fused luciferase gene constructs. The luciferase activity of the transfected cells increased with the glucose concentration in the medium, showing that the expression of the luciferase gene is regulated by SLCP(GL) , which can dissociate from DNA in a glucose concentration-dependent manner. Therefore, we demonstrated that SLCP(GL) functions as a glucose-sensitive transcriptional regulator in mammalian cells. These results reveal the possibility of developing an SBP-based regulator as a probe of intracellular sensing and gene regulation system for mammalian cells in response to a desired ligands depending on the SBP ligand specificity. Copyright © 2010 Wiley Periodicals, Inc.

  8. Heat shock protein 70 protects PC12 cells against ischemia-hypoxia/reoxygenation by maintaining intracellular Ca2+ homeostasis

    Directory of Open Access Journals (Sweden)

    Yuan Liu

    2016-01-01

    Full Text Available Heat shock protein 70 (HSP70 maintains Ca2+ homeostasis in PC12 cells, which may protect against apoptosis; however, the mechanisms of neuroprotection are unclear. Therefore, in this study, we examined Ca2+ levels in PC12 cells transfected with an exogenous lentiviral HSP70 gene expression construct, and we subsequently subjected the cells to ischemia-hypoxia/reoxygenation injury. HSP70 overexpression increased neuronal viability and ATPase activity, and it decreased cellular reactive oxygen species levels and intracellular Ca2+ concentration after hypoxia/reoxygenation. HSP70 overexpression enhanced the protein and mRNA expression levels of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA, but it decreased the protein and mRNA levels of inositol 1,4,5-trisphosphate receptor (IP3R, thereby leading to decreased intracellular Ca2+ concentration after ischemia-hypoxia/reoxygenation. These results suggest that exogenous HSP70 protects against ischemia-hypoxia/reoxygenation injury, at least in part, by maintaining cellular Ca2+ homeostasis, by upregulating SERCA expression and by downregulating IP3R expression.

  9. Inhibition of protein degradation induces apoptosis through a microtubule-associated protein 1 light chain 3-mediated activation of caspase-8 at intracellular membranes.

    Science.gov (United States)

    Pan, Ji-An; Ullman, Erica; Dou, Zhixun; Zong, Wei-Xing

    2011-08-01

    The accumulation of damaged or misfolded proteins, if unresolved, can lead to a detrimental consequence within cells termed proteotoxicity. Since cancerous cells often display elevated protein synthesis and by-product disposal, inhibition of the protein degradation pathways is an emerging approach for cancer therapy. However, the molecular mechanism underlying proteotoxicity remains largely unclear. We show here that inhibition of proteasomal degradation results in an increased oligomerization and activation of caspase-8 on the cytosolic side of intracellular membranes. This enhanced caspase-8 oligomerization and activation are promoted through its interaction with the ubiquitin-binding protein SQSTM1/p62 and the microtubule-associated protein light chain 3 (LC3), which are enriched at intracellular membranes in response to proteotoxic stress. Silencing LC3 by shRNA, or the LC3 mutants defective in membrane localization or p62 interaction fail to induce caspase-8 activation and apoptosis. Our results unveiled a previously unknown mechanism through which disruption of protein homeostasis induces caspase-8 oligomerization, activation, and apoptosis.

  10. Transmembrane protein OSTA-1 shapes sensory cilia morphology via regulation of intracellular membrane trafficking in C. elegans.

    Science.gov (United States)

    Olivier-Mason, Anique; Wojtyniak, Martin; Bowie, Rachel V; Nechipurenko, Inna V; Blacque, Oliver E; Sengupta, Piali

    2013-04-01

    The structure and function of primary cilia are critically dependent on intracellular trafficking pathways that transport ciliary membrane and protein components. The mechanisms by which these trafficking pathways are regulated are not fully characterized. Here we identify the transmembrane protein OSTA-1 as a new regulator of the trafficking pathways that shape the morphology and protein composition of sensory cilia in C. elegans. osta-1 encodes an organic solute transporter alpha-like protein, mammalian homologs of which have been implicated in membrane trafficking and solute transport, although a role in regulating cilia structure has not previously been demonstrated. We show that mutations in osta-1 result in altered ciliary membrane volume, branch length and complexity, as well as defects in localization of a subset of ciliary transmembrane proteins in different sensory cilia types. OSTA-1 is associated with transport vesicles, localizes to a ciliary compartment shown to house trafficking proteins, and regulates both retrograde and anterograde flux of the endosome-associated RAB-5 small GTPase. Genetic epistasis experiments with sensory signaling, exocytic and endocytic proteins further implicate OSTA-1 as a crucial regulator of ciliary architecture via regulation of cilia-destined trafficking. Our findings suggest that regulation of transport pathways in a cell type-specific manner contributes to diversity in sensory cilia structure and might allow dynamic remodeling of ciliary architecture via multiple inputs.

  11. Nesfatin-1 induces the phosphorylation levels of cAMP response element-binding protein for intracellular signaling in a neural cell line.

    Directory of Open Access Journals (Sweden)

    Emi Ishida

    Full Text Available Nesfatin-1 is a novel anorexic peptide that reduces the food intake of rodents when administered either intraventricularly or intraperitoneally. However, the molecular mechanism of intracellular signaling via Nesfatin-1 is yet to be resolved. In the current study, we investigated the ability of different neuronal cell lines to respond to Nesfatin-1 and further elucidated the signal transduction pathway of Nesfatin-1. To achieve this, we transfected several cell lines with various combinations of reporter vectors containing different kinds of response elements and performed reporter assays with Nesfatin-1, its active midsegment encoding 30 amino acid residues (M30 and M30-derived mutants. Notably, we found that both Nesfatin-1 as well as M30, significantly increased cAMP response element (CRE reporter activity in a mouse neuroblastoma cell line, NB41A3. An antagonist of Melanocortin 3/4 receptor, SHU9119, aborted the promoter activity, and a mutant M30, which exerts no anorexic effect in vivo did not induce the CRE reporter activity in NB41A3 cells. Western blotting analyses revealed that Nesfatin-1 and M30 significantly increased the phosphorylation levels of CRE-binding protein (CREB, without altering the intracellular cAMP levels. Further, our study showed that a mitogen-activated protein kinase (MAPK kinase inhibitor and an L-type Calcium (Ca(2+ channel blocker abolished the M30-induced CREB phosphorylation. Furthermore, the radio-receptor assay revealed that (125I-Nesfatin-1 binds in a saturable fashion to the membrane fractions of the mouse hypothalamus and NB41A3 cells, with Kd values of 0.79 nM and 0.17 nM, respectively. Collectively, our findings indicate the presence of a Nesfatin-1-specific receptor on the cell surface of NB41A3 cells and mouse hypothalamus. Our study highlights that Nesfatin-1, via its receptor, induces the phosphorylation of CREB, thus activating the intracellular signaling cascade in neurons.

  12. Sendai Virus C Protein Plays a Role in Restricting PKR Activation by Limiting the Generation of Intracellular Double-Stranded RNA▿

    OpenAIRE

    Takeuchi, Kenji; Komatsu, Takayuki; Kitagawa, Yoshinori; Sada, Kiyonao; Gotoh, Bin

    2008-01-01

    Sendai virus (SeV) C protein is a multifunctional protein that plays important roles in regulating viral genome replication and transcription, antagonizing the host interferon system, suppressing virus-induced apoptosis, and facilitating virus assembly and budding. We here report a novel role of SeV C protein, the limitation of double-stranded RNA (dsRNA) generation for maintaining the rate of protein synthesis in infected cells. It was found that the intracellular protein synthesis rate was ...

  13. HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways

    Science.gov (United States)

    Guo, Fang; Zhao, Qiong; Cheng, Junjun; Qi, Yonghe; Su, Qing; Wei, Lai; Li, Wenhui; Chang, Jinhong

    2017-01-01

    Hepatitis B virus (HBV) core protein assembles viral pre-genomic (pg) RNA and DNA polymerase into nucleocapsids for reverse transcriptional DNA replication to take place. Several chemotypes of small molecules, including heteroaryldihydropyrimidines (HAPs) and sulfamoylbenzamides (SBAs), have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or “empty” capsids devoid of pre-genomic RNA and viral DNA polymerase. Interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that HAPs and SBAs differentially modulate the biosynthesis of covalently closed circular (ccc) DNA from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded DNA-containing progeny nucleocapsids in the cytoplasm. Specifically, the mistimed cuing of nucleocapsid uncoating prevents cccDNA formation during de novo infection of hepatocytes, while transiently accelerating cccDNA synthesis from cytoplasmic progeny nucleocapsids. Our studies indicate that elongation of positive-stranded DNA induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind CpAMs and triggers its disassembly. Understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccDNA synthesis and cure chronic hepatitis B. PMID:28945802

  14. Expression and intracellular localization of TBC1D9, a Rab GTPase-accelerating protein, in mouse testes.

    Science.gov (United States)

    Nakamura, Yutaka; Asano, Atsushi; Hosaka, Yoshinao; Takeuchi, Takashi; Iwanaga, Toshihiko; Yamano, Yoshiaki

    2015-01-01

    Membrane trafficking in male germ cells contributes to their development via cell morphological changes and acrosome formation. TBC family proteins work as Rab GTPase accelerating proteins (GAPs), which negatively regulate Rab proteins, to mediate membrane trafficking. In this study, we analyzed the expression of a Rab GAP, TBC1D9, in mouse organs and the intracellular localization of the gene products. Tbc1d9 showed abundant expression in adult mice testis. We found that the Tbc1d9 mRNA was expressed in primary and secondary spermatocytes, and that the TBC1D9 protein was expressed in spermatocytes and round spermatids. In 293T cells, TBC1D9-GFP proteins were localized in the endosome and Golgi apparatus. Compartments that were positive for the constitutive active mutants of Rab7 and Rab9 were also positive for TBC1D9 isoform 1. In addition, TBC1D9 proteins were associated with Rab7 and Rab9, respectively. These results indicate that TBC1D9 is expressed mainly in spermatocytes, and suggest that TBC1D9 regulates membrane trafficking pathways related to Rab9- or Rab7-positive vesicles.

  15. Intracellular localization of Saffold virus Leader (L) protein differs in Vero and HEp-2 cells.

    Science.gov (United States)

    Xu, Yishi; Victorio, Carla Bianca Luena; Ng, Qimei; Prabakaran, Mookkan; Tan, Yee-Joo; Chua, Kaw Bing

    2016-10-12

    The Saffold virus (SAFV) genome is translated as a single long polyprotein precursor and co-translationally cleaved to yield 12 separate viral proteins. Little is known about the activities of SAFV proteins although their homologs in other picornaviruses have already been described. To further support research on functions and activities of respective viral proteins, we investigated the spatio-temporal distribution of SAFV proteins in Vero and HEp-2 cells that had been either transfected with plasmids that express individual viral proteins or infected with live SAFV. Our results revealed that, with the exception of the Leader (L) protein, all viral proteins were localized in the cytoplasm at all the time points assayed. The L protein was found in the cytoplasm at an early time point but was subsequently translocated to the nucleus of HEp-2, but not Vero, cells. This was observed in both transfected and infected cells. Further mutational analysis of L protein revealed that Threonine 58 of the Ser/Thr-rich domain of L protein is crucial for protein trafficking between the cytoplasm and nucleus in HEp-2 cells. These findings contribute to a deeper understanding and stimulate investigation of the differetial cellular responses of HEp-2 cells in comparison to other mammalian cell lines during SAFV infection.

  16. miR-92a enhances recombinant protein productivity in CHO cells by increasing intracellular cholesterol levels.

    Science.gov (United States)

    Loh, Wan Ping; Yang, Yuansheng; Lam, Kong Peng

    2017-04-01

    MicroRNAs (miRNAs) have emerged as promising targets for engineering of CHO cell factories to enhance recombinant protein productivity. Manipulation of miRNA levels in CHO cells have been shown to improve product yield by increasing proliferation and specific productivity (qP), resisting apoptosis and enhancing oxidative metabolism. The authors previously demonstrated that over-expressing miR-92a results in increases in qP and titer of CHO-IgG cells. However, the mechanisms by which miR-92a enhances qP in CHO cells are still uninvestigated. Here, the authors report the identification of insig1, a regulator of cholesterol biosynthesis, as a target of miR-92a using computational prediction. Both transient and stable over-expression of miR-92a decreased the expression levels of insig1. Insig1 was further validated as a target of miR-92a using 3' UTR reporter assay. Intracellular cholesterol concentration of two high-producing miR-92a clones were significantly increased by ≈30% compared to the blank-transfected pool. Relative Golgi surface area was also found to be 18-26% higher in these clones. Our findings suggest that miR-92a may affect cholesterol metabolism by repressing insig1, resulting in raised intracellular cholesterol levels and Golgi volume and hence enhanced protein secretion. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Membrane-protein integration and the role of the translocation channel.

    Science.gov (United States)

    Rapoport, Tom A; Goder, Veit; Heinrich, Sven U; Matlack, Kent E S

    2004-10-01

    Most eukaryotic membrane proteins are integrated into the lipid bilayer during their synthesis at the endoplasmic reticulum (ER). Their integration occurs with the help of a protein-conducting channel formed by the heterotrimeric Sec61 membrane-protein complex. The crystal structure of an archaeal homolog of the complex suggests mechanisms that enable the channel to open across the membrane and to release laterally hydrophobic transmembrane segments of nascent membrane proteins into lipid. Many aspects of membrane-protein integration remain controversial and poorly understood, but new structural data provide testable hypotheses. We propose a model of how the channel recognizes transmembrane segments, orients them properly with respect to the plane of the membrane and releases them into lipid. We also discuss how the channel would prevent small molecules from crossing the lipid bilayer while it is integrating proteins.

  18. A census of membrane-bound and intracellular signal transduction proteins in bacteria: Bacterial IQ, extroverts and introverts

    Directory of Open Access Journals (Sweden)

    Galperin Michael Y

    2005-06-01

    Full Text Available Abstract Background Analysis of complete microbial genomes showed that intracellular parasites and other microorganisms that inhabit stable ecological niches encode relatively primitive signaling systems, whereas environmental microorganisms typically have sophisticated systems of environmental sensing and signal transduction. Results This paper presents results of a comprehensive census of signal transduction proteins – histidine kinases, methyl-accepting chemotaxis receptors, Ser/Thr/Tyr protein kinases, adenylate and diguanylate cyclases and c-di-GMP phosphodiesterases – encoded in 167 bacterial and archaeal genomes, sequenced by the end of 2004. The data have been manually checked to avoid false-negative and false-positive hits that commonly arise during large-scale automated analyses and compared against other available resources. The census data show uneven distribution of most signaling proteins among bacterial and archaeal phyla. The total number of signal transduction proteins grows approximately as a square of genome size. While histidine kinases are found in representatives of all phyla and are distributed according to the power law, other signal transducers are abundant in certain phylogenetic groups but virtually absent in others. Conclusion The complexity of signaling systems differs even among closely related organisms. Still, it usually can be correlated with the phylogenetic position of the organism, its lifestyle, and typical environmental challenges it encounters. The number of encoded signal transducers (or their fraction in the total protein set can be used as a measure of the organism's ability to adapt to diverse conditions, the 'bacterial IQ', while the ratio of transmembrane receptors to intracellular sensors can be used to define whether the organism is an 'extrovert', actively sensing the environmental parameters, or an 'introvert', more concerned about its internal homeostasis. Some of the microorganisms with the

  19. A census of membrane-bound and intracellular signal transduction proteins in bacteria: bacterial IQ, extroverts and introverts.

    Science.gov (United States)

    Galperin, Michael Y

    2005-06-14

    Analysis of complete microbial genomes showed that intracellular parasites and other microorganisms that inhabit stable ecological niches encode relatively primitive signaling systems, whereas environmental microorganisms typically have sophisticated systems of environmental sensing and signal transduction. This paper presents results of a comprehensive census of signal transduction proteins--histidine kinases, methyl-accepting chemotaxis receptors, Ser/Thr/Tyr protein kinases, adenylate and diguanylate cyclases and c-di-GMP phosphodiesterases--encoded in 167 bacterial and archaeal genomes, sequenced by the end of 2004. The data have been manually checked to avoid false-negative and false-positive hits that commonly arise during large-scale automated analyses and compared against other available resources. The census data show uneven distribution of most signaling proteins among bacterial and archaeal phyla. The total number of signal transduction proteins grows approximately as a square of genome size. While histidine kinases are found in representatives of all phyla and are distributed according to the power law, other signal transducers are abundant in certain phylogenetic groups but virtually absent in others. The complexity of signaling systems differs even among closely related organisms. Still, it usually can be correlated with the phylogenetic position of the organism, its lifestyle, and typical environmental challenges it encounters. The number of encoded signal transducers (or their fraction in the total protein set) can be used as a measure of the organism's ability to adapt to diverse conditions, the 'bacterial IQ', while the ratio of transmembrane receptors to intracellular sensors can be used to define whether the organism is an 'extrovert', actively sensing the environmental parameters, or an 'introvert', more concerned about its internal homeostasis. Some of the microorganisms with the highest IQ, including the current leader Wolinella succinogenes

  20. A Generic Protocol for Intracellular Expression of Recombinant Proteins in Bacillus subtilis.

    Science.gov (United States)

    Phan, Trang; Huynh, Phuong; Truong, Tuom; Nguyen, Hoang

    2017-01-01

    Bacillus subtilis (B. subtilis) is a potential and attractive host for the production of recombinant proteins. Different expression systems for B. subtilis have been developed recently, and various target proteins have been recombinantly synthesized and purified using this host. In this chapter, we introduce a generic protocol to express a recombinant protein in B. subtilis. It includes protocols for (1) using our typical expression vector (plasmid pHT254) to introduce a target gene, (2) transformation of the target vector into B. subtilis, and (3) evaluation of the actual expression of a recombinant protein.

  1. Intracellular expression of IRF9 Stat fusion protein overcomes the defective Jak-Stat signaling and inhibits HCV RNA replication

    Directory of Open Access Journals (Sweden)

    Balart Luis A

    2010-10-01

    Full Text Available Abstract Interferon alpha (IFN-α binds to a cell surface receptor that activates the Jak-Stat signaling pathway. A critical component of this pathway is the translocation of interferon stimulated gene factor 3 (a complex of three proteins Stat1, Stat2 and IRF9 to the nucleus to activate antiviral genes. A stable sub-genomic replicon cell line resistant to IFN-α was developed in which the nuclear translocation of Stat1 and Stat2 proteins was prevented due to the lack of phosphorylation; whereas the nuclear translocation of IRF9 protein was not affected. In this study, we sought to overcome defective Jak-Stat signaling and to induce an antiviral state in the IFN-α resistant replicon cell line by developing a chimera IRF9 protein fused with the trans activating domain (TAD of either a Stat1 (IRF9-S1C or Stat2 (IRF9-S2C protein. We show here that intracellular expression of fusion proteins using the plasmid constructs of either IRF9-S1C or IRF9-S2C, in the IFN-α resistant cells, resulted in an increase in Interferon Stimulated Response Element (ISRE luciferase promoter activity and significantly induced HLA-1 surface expression. Moreover, we show that transient transfection of IRF9-S1C or IRF9-S2C plasmid constructs into IFN-α resistant replicon cells containing sub-genomic HCV1b and HCV2a viruses resulted in an inhibition of viral replication and viral protein expression independent of IFN-α treatment. The results of this study indicate that the recombinant fusion proteins of IRF9-S1C, IRF9-S2C alone, or in combination, have potent antiviral properties against the HCV in an IFN-α resistant cell line with a defective Jak-Stat signaling.

  2. The ratio of intracellular CRY proteins determines the clock period length

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yang [College of Biological Sciences, China Agricultural University, Beijing 100083 (China); National Institute of Biological Sciences, Beijing 102206 (China); Xiong, Wei [National Institute of Biological Sciences, Beijing 102206 (China); School of Life Sciences, Peking University, Beijing 100871 (China); Zhang, Eric Erquan, E-mail: zhangerquan@nibs.ac.cn [National Institute of Biological Sciences, Beijing 102206 (China)

    2016-04-08

    Although a deficiency in CRY1 or CRY2 correlates with a shorter or longer circadian period, the regulation of CRY proteins in the circadian period has not been well studied. In this study, we found that both CRY1 and CRY2 were able to rescue oscillation in CRY null cells and that they displayed different periods. Furthermore, we demonstrated that protein nuclear import rates, not protein stability, regulate the period-length at the cellular level. Co-transfection of CRY1 and CRY2 in various ratios in the same cells gives rise to the predicted period length in a dose-dependent manner. Given the distinct characteristics of the C-terminal tails of the CRY1 and CRY2 proteins, our study addresses a long-standing hypothesis that the ratio of these two CRY molecules affects the clock period. - Highlights: • Rhythmic CRY2, like CRY1, in the correct CRY1 phase is sufficient to rescue clock oscillation in CRY null cells. • The short-period mammalian CRY2 protein is more stable than the CRY1 protein. • The N-terminal polypeptide of CRY2 contributes to its stability and Per2 repression, but it does not affect the period. • The C-terminal tails of CRYs regulate their protein stability and nuclear import, but the import rate governs the period. • The ratio, rather than the absolute amounts of CRY1 and CRY2 proteins, determines the period in mammalian cells.

  3. Probing intracellular motor protein activity using an inducible cargo trafficking assay

    NARCIS (Netherlands)

    L.C. Kapitein (Lukas); M.A. Schlager (Max); W.A. van der Zwan (Wouter); P. Wulf (Phebe); N. Keijzer (Nanda); C.C. Hoogenraad (Casper)

    2010-01-01

    textabstractAlthough purified cytoskeletal motor proteins have been studied extensively with the use of in vitro approaches, a generic approach to selectively probe actin and microtubule-based motor protein activity inside living cells is lacking. To examine specific motor activity inside living

  4. Dissecting the Wnt secretion pathway: key questions on the modification and intracellular trafficking of Wnt proteins

    NARCIS (Netherlands)

    Harterink, M.; Korswagen, H.C.

    2012-01-01

    The Wnt family of signalling proteins has essential functions in development and adult tissue homoeostasis throughout the animal kingdom. Although signalling cascades triggered by Wnt proteins have been extensively studied, much remains to be learned about how Wnts are produced and secreted. Over

  5. Intracellular localization and movement phenotypes of alfalfa mosaic virus movement protein mutants

    NARCIS (Netherlands)

    Huang, M.; Jongejan, L.; Zheng, H.; Zhang, L.; Bol, J. F.

    2001-01-01

    Thirteen mutations were introduced in the movement protein (MP) gene of Alfalfa mosaic virus (AMV) fused to the green fluorescent protein (GFP) gene and the mutant MP-GFP fusions were expressed transiently in tobacco protoplasts, tobacco suspension cells, and epidermal cells of tobacco leaves. In

  6. Modulation of firing and synaptic transmission of serotonergic neurons by intrinsic G protein-coupled receptors and ion channels

    Directory of Open Access Journals (Sweden)

    Takashi eMaejima

    2013-05-01

    Full Text Available Serotonergic neurons project to virtually all regions of the CNS and are consequently involved in many critical physiological functions such as mood, sexual behavior, feeding, sleep/wake cycle, memory, cognition, blood pressure regulation, breathing and reproductive success. Therefore serotonin release and serotonergic neuronal activity have to be precisely controlled and modulated by interacting brain circuits to adapt to specific emotional and environmental states. We will review the current knowledge about G protein-coupled receptors and ion channels involved in the regulation of serotonergic system, how their regulation is modulating the intrinsic activity of serotonergic neurons and its transmitter release and will discuss the latest methods for controlling the modulation of serotonin release and intracellular signaling in serotonergic neurons in vitro and in vivo.

  7. The cannabinoid agonist WIN55,212-2 increases intracellular calcium via CB1 receptor coupling to Gq/11 G proteins

    OpenAIRE

    Lauckner, Jane E.; Hille, Bertil; Mackie, Ken

    2005-01-01

    Central nervous system responses to cannabis are primarily mediated by CB1 receptors, which couple preferentially to Gi/o G proteins. Here, we used calcium photometry to monitor the effect of CB1 activation on intracellular calcium concentration. Perfusion with 5 μM CB1 aminoalkylindole agonist, WIN55,212-2 (WIN), increased intracellular calcium by several hundred nanomolar in human embryonic kidney 293 cells stably expressing CB1 and in cultured hippocampal neurons. The increase was blocked ...

  8. A General Method for Intracellular Protein Delivery through 'E-tag' Protein Engineering and Arginine Functionalized Gold Nanoparticles.

    Science.gov (United States)

    Mout, Rubul; Rotello, Vincent M

    2017-12-20

    In this protocol, we describe a method for direct cytosolic protein delivery that avoids endosomal entrapment of the delivered proteins. We achieved this by tagging the desired protein with an oligo glutamic acid tag (E-tag), and subsequently using carrier gold nanoparticles to deliver these E-tagged proteins. When E-tagged proteins and nanoparticles were mixed, they formed nanoassemblies, which got fused to cell membrane upon incubation and directly released the E-tagged protein into cell cytosol. We used this method to deliver a wide variety of proteins with different sizes, charges, and functions in various cell lines (Mout et al ., 2017). To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant E-tagged proteins). Laboratory-synthesis of gold nanoparticles has been previously described (Yang et al ., 2011). Desired E-tagged proteins can be cloned from the corresponding genes, and expressed and purified using standard laboratory procedures. We will use E-tagged green fluorescent protein (GFP) as a reference protein here. Users can simply insert an E-tag into their protein of interest, at either terminus. To achieve maximum delivery efficiency, we suggest users testing different length of E-tags. For example, we inserted E = 0 to 20 (E0 means no E-tag insertion, and E20 means 20 glutamic acids insertion in a row) to most of the proteins we tested, and screened for optimal E-tagged length for highest delivery efficiency. E10-tagged proteins gave us the highest delivery efficiency for most of the proteins (except for Cas9, where E20 tag showed highest delivery efficiency). Once these materials are ready, it takes about ~10 min to make the E-tagged protein and nanoparticle nanoassemblies, which are immediately used for delivery. Complete delivery (~100% for GFP-E10) is achieved in less than 3 h.

  9. Activation of PKA and Epac proteins by cyclic AMP depletes intracellular calcium stores and reduces calcium availability for vasoconstriction.

    Science.gov (United States)

    Cuíñas, Andrea; García-Morales, Verónica; Viña, Dolores; Gil-Longo, José; Campos-Toimil, Manuel

    2016-06-15

    We investigated the implication of PKA and Epac proteins in the endothelium-independent vasorelaxant effects of cyclic AMP (cAMP). Cytosolic Ca(2+) concentration ([Ca(2+)]c) was measured by fura-2 imaging in rat aortic smooth muscle cells (RASMC). Contraction-relaxation experiments were performed in rat aortic rings deprived of endothelium. In extracellular Ca(2+)-free solution, cAMP-elevating agents induced an increase in [Ca(2+)]c in RASMC that was reproduced by PKA and Epac activation and reduced after depletion of intracellular Ca(2+) reservoirs. Arginine-vasopressin (AVP)-evoked increase of [Ca(2+)]c and store-operated Ca(2+) entry (SOCE) were inhibited by cAMP-elevating agents, PKA or Epac activation in these cells. In aortic rings, the contractions induced by phenylephrine in absence of extracellular Ca(2+) were inhibited by cAMP-elevating agents, PKA or Epac activation. In these conditions, reintroduction of Ca(2+) induced a contraction that was inhibited by cAMP-elevating agents, an effect reduced by PKA inhibition and reproduced by PKA or Epac activators. Our results suggest that increased cAMP depletes intracellular, thapsigargin-sensitive Ca(2+) stores through activation of PKA and Epac in RASMC, thus reducing the amount of Ca(2+) released by IP3-generating agonists during the contraction of rat aorta. cAMP rise also inhibits the contraction induced by depletion of intracellular Ca(2+), an effect mediated by reduction of SOCE after PKA or Epac activation. Both effects participate in the cAMP-induced endothelium-independent vasorelaxation. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Fatty acid-binding proteins (FABPs) are intracellular carriers for Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD).

    Science.gov (United States)

    Elmes, Matthew W; Kaczocha, Martin; Berger, William T; Leung, KwanNok; Ralph, Brian P; Wang, Liqun; Sweeney, Joseph M; Miyauchi, Jeremy T; Tsirka, Stella E; Ojima, Iwao; Deutsch, Dale G

    2015-04-03

    Δ(9)-Tetrahydrocannabinol (THC) and cannabidiol (CBD) occur naturally in marijuana (Cannabis) and may be formulated, individually or in combination in pharmaceuticals such as Marinol or Sativex. Although it is known that these hydrophobic compounds can be transported in blood by albumin or lipoproteins, the intracellular carrier has not been identified. Recent reports suggest that CBD and THC elevate the levels of the endocannabinoid anandamide (AEA) when administered to humans, suggesting that phytocannabinoids target cellular proteins involved in endocannabinoid clearance. Fatty acid-binding proteins (FABPs) are intracellular proteins that mediate AEA transport to its catabolic enzyme fatty acid amide hydrolase (FAAH). By computational analysis and ligand displacement assays, we show that at least three human FABPs bind THC and CBD and demonstrate that THC and CBD inhibit the cellular uptake and catabolism of AEA by targeting FABPs. Furthermore, we show that in contrast to rodent FAAH, CBD does not inhibit the enzymatic actions of human FAAH, and thus FAAH inhibition cannot account for the observed increase in circulating AEA in humans following CBD consumption. Using computational molecular docking and site-directed mutagenesis we identify key residues within the active site of FAAH that confer the species-specific sensitivity to inhibition by CBD. Competition for FABPs may in part or wholly explain the increased circulating levels of endocannabinoids reported after consumption of cannabinoids. These data shed light on the mechanism of action of CBD in modulating the endocannabinoid tone in vivo and may explain, in part, its reported efficacy toward epilepsy and other neurological disorders. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Fatty Acid-binding Proteins (FABPs) Are Intracellular Carriers for Δ9-Tetrahydrocannabinol (THC) and Cannabidiol (CBD)*

    Science.gov (United States)

    Elmes, Matthew W.; Kaczocha, Martin; Berger, William T.; Leung, KwanNok; Ralph, Brian P.; Wang, Liqun; Sweeney, Joseph M.; Miyauchi, Jeremy T.; Tsirka, Stella E.; Ojima, Iwao; Deutsch, Dale G.

    2015-01-01

    Δ9-Tetrahydrocannabinol (THC) and cannabidiol (CBD) occur naturally in marijuana (Cannabis) and may be formulated, individually or in combination in pharmaceuticals such as Marinol or Sativex. Although it is known that these hydrophobic compounds can be transported in blood by albumin or lipoproteins, the intracellular carrier has not been identified. Recent reports suggest that CBD and THC elevate the levels of the endocannabinoid anandamide (AEA) when administered to humans, suggesting that phytocannabinoids target cellular proteins involved in endocannabinoid clearance. Fatty acid-binding proteins (FABPs) are intracellular proteins that mediate AEA transport to its catabolic enzyme fatty acid amide hydrolase (FAAH). By computational analysis and ligand displacement assays, we show that at least three human FABPs bind THC and CBD and demonstrate that THC and CBD inhibit the cellular uptake and catabolism of AEA by targeting FABPs. Furthermore, we show that in contrast to rodent FAAH, CBD does not inhibit the enzymatic actions of human FAAH, and thus FAAH inhibition cannot account for the observed increase in circulating AEA in humans following CBD consumption. Using computational molecular docking and site-directed mutagenesis we identify key residues within the active site of FAAH that confer the species-specific sensitivity to inhibition by CBD. Competition for FABPs may in part or wholly explain the increased circulating levels of endocannabinoids reported after consumption of cannabinoids. These data shed light on the mechanism of action of CBD in modulating the endocannabinoid tone in vivo and may explain, in part, its reported efficacy toward epilepsy and other neurological disorders. PMID:25666611

  12. Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

    International Nuclear Information System (INIS)

    Zhao Weihong; Zhong Li; Wu Jianqing; Chen Linyuan; Qing Keyun; Weigel-Kelley, Kirsten A.; Larsen, Steven H.; Shou Weinian; Warrington, Kenneth H.; Srivastava, Arun

    2006-01-01

    We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by ∼25-fold in WT MEFs, but only by ∼4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency ∼23-fold in WT MEFs, but only ∼4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, ∼59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only ∼28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene

  13. Plant VAP27 proteins: domain characterization, intracellular localization and role in plant development.

    Science.gov (United States)

    Wang, Pengwei; Richardson, Christine; Hawkins, Timothy J; Sparkes, Imogen; Hawes, Chris; Hussey, Patrick J

    2016-06-01

    The endoplasmic reticulum (ER) is connected to the plasma membrane (PM) through the plant-specific NETWORKED protein, NET3C, and phylogenetically conserved vesicle-associated membrane protein-associated proteins (VAPs). Ten VAP homologues (VAP27-1 to 27-10) can be identified in the Arabidopsis genome and can be divided into three clades. Representative members from each clade were tagged with fluorescent protein and expressed in Nicotiana benthamiana. Proteins from clades I and III localized to the ER as well as to ER/PM contact sites (EPCSs), whereas proteins from clade II were found only at the PM. Some of the VAP27-labelled EPCSs localized to plasmodesmata, and we show that the mobility of VAP27 at EPCSs is influenced by the cell wall. EPCSs closely associate with the cytoskeleton, but their structure is unaffected when the cytoskeleton is removed. VAP27-labelled EPCSs are found in most cell types in Arabidopsis, with the exception of cells in early trichome development. Arabidopsis plants expressing VAP27-GFP fusions exhibit pleiotropic phenotypes, including defects in root hair morphogenesis. A similar effect is also observed in plants expressing VAP27 RNAi. Taken together, these data indicate that VAP27 proteins used at EPCSs are essential for normal ER-cytoskeleton interaction and for plant development. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  14. Conformational landscape of an amyloid intra-cellular domain and Landau-Ginzburg-Wilson paradigm in protein dynamics

    International Nuclear Information System (INIS)

    Dai, Jin; He, Jianfeng; Niemi, Antti J.

    2016-01-01

    The Landau-Ginzburg-Wilson paradigm is proposed as a framework, to investigate the conformational landscape of intrinsically unstructured proteins. A universal Cα-trace Landau free energy is deduced from general symmetry considerations, with the ensuing all-atom structure modeled using publicly available reconstruction programs Pulchra and Scwrl. As an example, the conformational stability of an amyloid precursor protein intra-cellular domain (AICD) is inspected; the reference conformation is the crystallographic structure with code 3DXC in Protein Data Bank (PDB) that describes a heterodimer of AICD and a nuclear multi-domain adaptor protein Fe65. Those conformations of AICD that correspond to local or near-local minima of the Landau free energy are identified. For this, the response of the original 3DXC conformation to variations in the ambient temperature is investigated, using the Glauber algorithm. The conclusion is that in isolation the AICD conformation in 3DXC must be unstable. A family of degenerate conformations that minimise the Landau free energy is identified, and it is proposed that the native state of an isolated AICD is a superposition of these conformations. The results are fully in line with the presumed intrinsically unstructured character of isolated AICD and should provide a basis for a systematic analysis of AICD structure in future NMR experiments.

  15. Conformational landscape of an amyloid intra-cellular domain and Landau-Ginzburg-Wilson paradigm in protein dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Jin; He, Jianfeng, E-mail: Antti.Niemi@physics.uu.se, E-mail: hjf@bit.edu.cn [School of Physics, Beijing Institute of Technology, Beijing 100081 (China); Niemi, Antti J., E-mail: Antti.Niemi@physics.uu.se, E-mail: hjf@bit.edu.cn [School of Physics, Beijing Institute of Technology, Beijing 100081 (China); Department of Physics and Astronomy, Uppsala University, P.O. Box 803, S-75108 Uppsala (Sweden); Laboratoire de Mathematiques et Physique Theorique CNRS UMR 6083, Fédération Denis Poisson, Université de Tours, Parc de Grandmont, F37200 Tours (France)

    2016-07-28

    The Landau-Ginzburg-Wilson paradigm is proposed as a framework, to investigate the conformational landscape of intrinsically unstructured proteins. A universal Cα-trace Landau free energy is deduced from general symmetry considerations, with the ensuing all-atom structure modeled using publicly available reconstruction programs Pulchra and Scwrl. As an example, the conformational stability of an amyloid precursor protein intra-cellular domain (AICD) is inspected; the reference conformation is the crystallographic structure with code 3DXC in Protein Data Bank (PDB) that describes a heterodimer of AICD and a nuclear multi-domain adaptor protein Fe65. Those conformations of AICD that correspond to local or near-local minima of the Landau free energy are identified. For this, the response of the original 3DXC conformation to variations in the ambient temperature is investigated, using the Glauber algorithm. The conclusion is that in isolation the AICD conformation in 3DXC must be unstable. A family of degenerate conformations that minimise the Landau free energy is identified, and it is proposed that the native state of an isolated AICD is a superposition of these conformations. The results are fully in line with the presumed intrinsically unstructured character of isolated AICD and should provide a basis for a systematic analysis of AICD structure in future NMR experiments.

  16. Genetic Code Expansion- and Click Chemistry-Based Site-Specific Protein Labeling for Intracellular DNA-PAINT Imaging.

    Science.gov (United States)

    Nikić-Spiegel, Ivana

    2018-01-01

    Super-resolution microscopy allows imaging of cellular structures at nanometer resolution. This comes with a demand for small labels which can be attached directly to the structures of interest. In the context of protein labeling, one way to achieve this is by using genetic code expansion (GCE) and click chemistry. With GCE, small labeling handles in the form of noncanonical amino acids (ncAAs) are site-specifically introduced into a target protein. In a subsequent step, these amino acids can be directly labeled with small organic dyes by click chemistry reactions. Click chemistry labeling can also be combined with other methods, such as DNA-PAINT in which a "clickable" oligonucleotide is first attached to the ncAA-bearing target protein and then labeled with complementary fluorescent oligonucleotides. This protocol will cover both aspects: I describe (1) how to encode ncAAs and perform intracellular click chemistry-based labeling with an improved GCE system for eukaryotic cells and (2) how to combine click chemistry-based labeling with DNA-PAINT super-resolution imaging. As an example, I show click-PAINT imaging of vimentin and low-abundance nuclear protein, nucleoporin 153.

  17. Specific presence of intracellular citrullinated proteins in rheumatoid arthritis synovium: relevance to antifilaggrin autoantibodies

    NARCIS (Netherlands)

    Baeten, D.; Peene, I.; Union, A.; Meheus, L.; Sebbag, M.; Serre, G.; Veys, E. M.; de Keyser, F.

    2001-01-01

    OBJECTIVE: To investigate the presence of citrullinated proteins in the synovial membrane of patients with rheumatoid arthritis (RA) and controls, and to analyze a possible relationship with antifilaggrin autoantibody (AFA) reactivity. METHODS: Synovial biopsy samples were obtained from 88

  18. Intracellular localization of group 3 LEA proteins in embryos of Artemia franciscana.

    Science.gov (United States)

    Boswell, Leaf C; Hand, Steven C

    2014-12-01

    Late embryogenesis abundant (LEA) proteins are accumulated by anhydrobiotic organisms in response to desiccation and improve survivorship during water stress. In this study we provide the first direct evidence for the subcellular localizations of AfrLEA2 and AfrLEA3m (and its subforms) in anhydrobiotic embryos of Artemia franciscana. Immunohistochemistry shows AfrLEA2 to reside in the cytoplasm and nucleus, and the four AfrLEA3m proteins to be localized to the mitochondrion. Cellular locations are supported by Western blots of mitochondrial, nuclear and cytoplasmic fractions. The presence of LEA proteins in multiple subcellular compartments of A. franciscana embryos suggests the need to protect biological structures in many areas of a cell in order for an organism to survive desiccation stress, and may explain in part why a multitude of different LEA proteins are expressed by a single organism. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. A Toxoplasma gondii protein with homology to intracellular type Na+/H+ exchangers is important for osmoregulation and invasion

    International Nuclear Information System (INIS)

    Francia, Maria E.; Wicher, Sarah; Pace, Douglas A.; Sullivan, Jack; Moreno, Silvia N.J.; Arrizabalaga, Gustavo

    2011-01-01

    The obligate intracellular parasite Toxoplasma gondii is exposed to a variety of physiological conditions while propagating in an infected organism. The mechanisms by which Toxoplasma overcomes these dramatic changes in its environment are not known. In yeast and plants, ion detoxification and osmotic regulation are controlled by vacuolar compartments. A novel compartment named the plant-like vacuole or vacuolar compartment (PLV/VAC) has recently been described in T.gondii, which could potentially protect extracellular tachyzoites against salt and other ionic stresses. Here, we report the molecular characterization of the vacuolar type Na + /H + exchanger in T. gondii, TgNHE3, and its co-localization with the PLV/VAC proton-pyrophosphatase (TgVP1). We have created a TgNHE3 knockout strain, which is more sensitive to hyperosmotic shock and toxic levels of sodium, possesses a higher intracellular Ca 2+ concentration [Ca 2+ ] i , and exhibits a reduced host invasion efficiency. The defect in invasion correlates with a measurable reduction in the secretion of the adhesin TgMIC2. Overall, our results suggest that the PLV/VAC has functions analogous to those of the vacuolar compartments of plants and yeasts, providing the parasite with a mechanism to resist ionic fluctuations and, potentially, regulate protein trafficking.

  20. Phycodnavirus potassium ion channel proteins question the virus molecular piracy hypothesis.

    Science.gov (United States)

    Hamacher, Kay; Greiner, Timo; Ogata, Hiroyuki; Van Etten, James L; Gebhardt, Manuela; Villarreal, Luis P; Cosentino, Cristian; Moroni, Anna; Thiel, Gerhard

    2012-01-01

    Phycodnaviruses are large dsDNA, algal-infecting viruses that encode many genes with homologs in prokaryotes and eukaryotes. Among the viral gene products are the smallest proteins known to form functional K(+) channels. To determine if these viral K(+) channels are the product of molecular piracy from their hosts, we compared the sequences of the K(+) channel pore modules from seven phycodnaviruses to the K(+) channels from Chlorella variabilis and Ectocarpus siliculosus, whose genomes have recently been sequenced. C. variabilis is the host for two of the viruses PBCV-1 and NY-2A and E. siliculosus is the host for the virus EsV-1. Systematic phylogenetic analyses consistently indicate that the viral K(+) channels are not related to any lineage of the host channel homologs and that they are more closely related to each other than to their host homologs. A consensus sequence of the viral channels resembles a protein of unknown function from a proteobacterium. However, the bacterial protein lacks the consensus motif of all K(+) channels and it does not form a functional channel in yeast, suggesting that the viral channels did not come from a proteobacterium. Collectively, our results indicate that the viruses did not acquire their K(+) channel-encoding genes from their current algal hosts by gene transfer; thus alternative explanations are required. One possibility is that the viral genes arose from ancient organisms, which served as their hosts before the viruses developed their current host specificity. Alternatively the viral proteins could be the origin of K(+) channels in algae and perhaps even all cellular organisms.

  1. Phycodnavirus potassium ion channel proteins question the virus molecular piracy hypothesis.

    Directory of Open Access Journals (Sweden)

    Kay Hamacher

    Full Text Available Phycodnaviruses are large dsDNA, algal-infecting viruses that encode many genes with homologs in prokaryotes and eukaryotes. Among the viral gene products are the smallest proteins known to form functional K(+ channels. To determine if these viral K(+ channels are the product of molecular piracy from their hosts, we compared the sequences of the K(+ channel pore modules from seven phycodnaviruses to the K(+ channels from Chlorella variabilis and Ectocarpus siliculosus, whose genomes have recently been sequenced. C. variabilis is the host for two of the viruses PBCV-1 and NY-2A and E. siliculosus is the host for the virus EsV-1. Systematic phylogenetic analyses consistently indicate that the viral K(+ channels are not related to any lineage of the host channel homologs and that they are more closely related to each other than to their host homologs. A consensus sequence of the viral channels resembles a protein of unknown function from a proteobacterium. However, the bacterial protein lacks the consensus motif of all K(+ channels and it does not form a functional channel in yeast, suggesting that the viral channels did not come from a proteobacterium. Collectively, our results indicate that the viruses did not acquire their K(+ channel-encoding genes from their current algal hosts by gene transfer; thus alternative explanations are required. One possibility is that the viral genes arose from ancient organisms, which served as their hosts before the viruses developed their current host specificity. Alternatively the viral proteins could be the origin of K(+ channels in algae and perhaps even all cellular organisms.

  2. The jellyfish green fluorescent protein: a new tool for studying ion channel expression and function.

    Science.gov (United States)

    Marshall, J; Molloy, R; Moss, G W; Howe, J R; Hughes, T E

    1995-02-01

    Two methods are described for using the jellyfish green fluorescent protein (GFP) as a reporter gene for ion channel expression. GFP fluorescence can be used to identify the transfected cells, and to estimate the relative levels of ion channel expression, in cotransfection experiments. A GFP-NMDAR1 chimera can be constructed that produces a functional, fluorescent receptor subunit. These methods should facilitate studies of ion channel expression, localization, and processing.

  3. Involvement of TRPV3 and TRPM8 ion channel proteins in induction of mammalian cold-inducible proteins.

    Science.gov (United States)

    Fujita, Takanori; Liu, Yu; Higashitsuji, Hiroaki; Itoh, Katsuhiko; Shibasaki, Koji; Fujita, Jun; Nishiyama, Hiroyuki

    2018-01-01

    Cold-inducible RNA-binding protein (CIRP), RNA-binding motif protein 3 (RBM3) and serine and arginine rich splicing factor 5 (SRSF5) are RNA-binding proteins that are transcriptionally upregulated in response to moderately low temperatures and a variety of cellular stresses in mammalian cells. Induction of these cold-inducible proteins (CIPs) is dependent on transient receptor potential (TRP) V4 channel protein, but seems independent of its ion channel activity. We herein report that in addition to TRPV4, TRPV3 and TRPM8 are necessary for the induction of CIPs. We established cell lines from the lung of TRPV4-knockout (KO) mouse, and observed induction of CIPs in them by western blot analysis. A TRPV4 antagonist RN1734 suppressed the induction in wild-type mouse cells, but not in TRPV4-KO cells. A TRPV3 channel blocker S408271 and a TRPM8 channel blocker AMTB as well as siRNAs against TRPV3 and TRPM8 suppressed the CIP induction in mouse TRPV4-KO cells and human U-2 OS cells. A TRPV3 channel agonist 2-APB induced CIP expression, but camphor did not. Neither did a TRPM8 channel agonist WS-12. These results suggest that TRPV4, TRPV3 and TRPM8 proteins, but not their ion channel activities are necessary for the induction of CIPs at 32 °C. Identification of proteins that differentially interact with these TRP channels at 37 °C and 32 °C would help elucidate the underlying mechanisms of CIP induction by hypothermia. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  4. pH-sensitive degradable nanoparticles for highly efficient intracellular delivery of exogenous protein

    Directory of Open Access Journals (Sweden)

    Xu D

    2013-09-01

    Full Text Available Dan Xu,1 Fei Wu,1 Yinghui Chen,2,* Liangming Wei,3,* Weien Yuan1,* 1School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 2Department of Neurology, Jinshan Hospital, Fudan University, Shanghai, 3Key Laboratory for Thin Film and Microfabrication of the Ministry of Education, Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, Shanghai, People's Republic of China*These authors contributed equally to this workBackground: Encapsulating exogenous proteins into a nanosized particulate system for delivery into cells is a great challenge. To address this issue, we developed a novel nanoparticle delivery method that differs from the nanoparticles reported to date because its core was composed of cross-linked dextran glassy nanoparticles which had pH in endosome-responsive environment and the protein was loaded in the core of cross-linked dextran glassy nanoparticles.Methods: In this study, dextran in a poly(ethylene glycol aqueous two-phase system created a different chemical environment in which proteins were encapsulated very efficiently (84.3% and 89.6% for enhanced green fluorescent protein and bovine serum albumin, respectively by thermodynamically favored partition. The structures of the nanoparticles were confirmed by confocal laser scanning microscopy and scanning electron microscopy.Results: The nanoparticles had a normal size distribution and a mean diameter of 186 nm. MTT assays showed that the nanoparticles were nontoxic up to a concentration of 2000 µg/mL in human hepatocarcinoma cell line SMMC-7721, HeLa, and BRL-3A cells. Of note, confocal laser scanning microscopy studies showed that nanoparticles loaded with fluorescein isothiocyanate-bovine serum albumin were efficiently delivered and released proteins into the cytoplasm of HeLa cells. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that nanoparticles with a functional protein (apoptin efficiently induced

  5. Complete genome sequence and intracellular protein localization of Datura yellow vein nucleorhabdovirus.

    Science.gov (United States)

    Dietzgen, Ralf G; Innes, David J; Bejerman, Nicolas

    2015-07-02

    A limited number of plant rhabdovirus genomes have been fully sequenced, making taxonomic classification, evolutionary analysis and molecular characterization of this virus group difficult. We have for the first time determined the complete genome sequence of 13,188 nucleotides of Datura yellow vein nucleorhabdovirus (DYVV). DYVV genome organization resembles that of its closest relative, Sonchus yellow net virus (SYNV), with six ORFs in antigenomic orientation, separated by highly conserved intergenic regions and flanked by complementary 3' leader and 5' trailer sequences. As is typical for nucleorhabdoviruses, all viral proteins, except the glycoprotein, which is targeted to the endoplasmic reticulum, are localized to the nucleus. Nucleocapsid (N) protein, matrix (M) protein and polymerase, as components of nuclear viroplasms during replication, have predicted strong canonical nuclear localization signals, and N and M proteins exclusively localize to the nucleus when transiently expressed as GFP fusions. As in all nucleorhabdoviruses studied so far, N and phosphoprotein P interact when co-expressed, significantly increasing P nuclear localization in the presence of N protein. This research adds to the list of complete genomes of plant-infecting rhabdoviruses, provides molecular tools for further characterization and supports classification of DYVV as a nucleorhabdovirus closely related to but with some distinct differences from SYNV. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Central functions of bicarbonate in S-type anion channel activation and OST1 protein kinase in CO 2 signal transduction in guard cell

    KAUST Repository

    Xue, Shaowu

    2011-03-18

    Plants respond to elevated CO(2) via carbonic anhydrases that mediate stomatal closing, but little is known about the early signalling mechanisms following the initial CO(2) response. It remains unclear whether CO(2), HCO(3)(-) or a combination activates downstream signalling. Here, we demonstrate that bicarbonate functions as a small-molecule activator of SLAC1 anion channels in guard cells. Elevated intracellular [HCO(3)(-)](i) with low [CO(2)] and [H(+)] activated S-type anion currents, whereas low [HCO(3)(-)](i) at high [CO(2)] and [H(+)] did not. Bicarbonate enhanced the intracellular Ca(2+) sensitivity of S-type anion channel activation in wild-type and ht1-2 kinase mutant guard cells. ht1-2 mutant guard cells exhibited enhanced bicarbonate sensitivity of S-type anion channel activation. The OST1 protein kinase has been reported not to affect CO(2) signalling. Unexpectedly, OST1 loss-of-function alleles showed strongly impaired CO(2)-induced stomatal closing and HCO(3)(-) activation of anion channels. Moreover, PYR/RCAR abscisic acid (ABA) receptor mutants slowed but did not abolish CO(2)/HCO(3)(-) signalling, redefining the convergence point of CO(2) and ABA signalling. A new working model of the sequence of CO(2) signalling events in gas exchange regulation is presented.

  7. Improved intracellular delivery of glucocerebrosidase mediated by the HIV-1 TAT protein transduction domain

    International Nuclear Information System (INIS)

    Lee, Kyun Oh; Luu, Nga; Kaneski, Christine R.; Schiffmann, Raphael; Brady, Roscoe O.; Murray, Gary J.

    2005-01-01

    Enzyme replacement therapy (ERT) for Gaucher disease designed to target glucocerebrosidase (GC) to macrophages via mannose-specific endocytosis is very effective in reversing hepatosplenomegaly, and normalizing hematologic parameters but is less effective in improving bone and lung involvement and ineffective in brain. Recombinant GCs containing an in-frame fusion to the HIV-1 trans-activator protein transduction domain (TAT) were expressed in eukaryotic cells in order to obtain active, normally glycosylated GC fusion proteins for enzyme uptake studies. Despite the absence of mannose-specific endocytic receptors on the plasma membranes of various fibroblasts, the recombinant GCs with C-terminal TAT fusions were readily internalized by these cells. Immunofluorescent confocal microscopy demonstrated the recombinant TAT-fusion proteins with a mixed endosomal and lysosomal localization. Thus, TAT-modified GCs represent a novel strategy for a new generation of therapeutic enzymes for ERT for Gaucher disease

  8. Structure of the virulence-associated protein VapD from the intracellular pathogen Rhodococcus equi

    International Nuclear Information System (INIS)

    Whittingham, Jean L.; Blagova, Elena V.; Finn, Ciaran E.; Luo, Haixia; Miranda-CasoLuengo, Raúl; Turkenburg, Johan P.; Leech, Andrew P.; Walton, Paul H.; Murzin, Alexey G.; Meijer, Wim G.; Wilkinson, Anthony J.

    2014-01-01

    VapD is one of a set of highly homologous virulence-associated proteins from the multi-host pathogen Rhodococcus equi. The crystal structure reveals an eight-stranded β-barrel with a novel fold and a glycine rich ‘bald’ surface. Rhodococcus equi is a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity of R. equi to divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vap proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as determined by multi-angle laser light scattering. The structure reveals an elliptical, compact eight-stranded β-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-β-d-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis suggests that the β-barrel structure is preceded by a natively disordered region at the N-terminus. Sequence comparisons indicate that the core folds of the other plasmid-encoded virulence-associated proteins from R. equi strains are similar to that of VapD. It is further shown that sequences encoding putative R. equi Vap-like proteins occur in diverse bacterial species. Finally, the functional implications of the structure are discussed in the light of the unique structural features of VapD and its partial structural similarity to other β-barrel proteins

  9. Structure of the virulence-associated protein VapD from the intracellular pathogen Rhodococcus equi

    Energy Technology Data Exchange (ETDEWEB)

    Whittingham, Jean L.; Blagova, Elena V. [University of York, Heslington, York YO10 5DD (United Kingdom); Finn, Ciaran E.; Luo, Haixia; Miranda-CasoLuengo, Raúl [University College Dublin, Dublin (Ireland); Turkenburg, Johan P.; Leech, Andrew P.; Walton, Paul H. [University of York, Heslington, York YO10 5DD (United Kingdom); Murzin, Alexey G. [MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH (United Kingdom); Meijer, Wim G. [University College Dublin, Dublin (Ireland); Wilkinson, Anthony J., E-mail: tony.wilkinson@york.ac.uk [University of York, Heslington, York YO10 5DD (United Kingdom)

    2014-08-01

    VapD is one of a set of highly homologous virulence-associated proteins from the multi-host pathogen Rhodococcus equi. The crystal structure reveals an eight-stranded β-barrel with a novel fold and a glycine rich ‘bald’ surface. Rhodococcus equi is a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity of R. equi to divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vap proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as determined by multi-angle laser light scattering. The structure reveals an elliptical, compact eight-stranded β-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-β-d-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis suggests that the β-barrel structure is preceded by a natively disordered region at the N-terminus. Sequence comparisons indicate that the core folds of the other plasmid-encoded virulence-associated proteins from R. equi strains are similar to that of VapD. It is further shown that sequences encoding putative R. equi Vap-like proteins occur in diverse bacterial species. Finally, the functional implications of the structure are discussed in the light of the unique structural features of VapD and its partial structural similarity to other β-barrel proteins.

  10. Generation of GFP Native Protein for Detection of Its Intracellular Uptake by Cell-Penetrating Peptides.

    Science.gov (United States)

    Kadkhodayan, S; Sadat, S M; Irani, S; Fotouhi, F; Bolhassani, A

    2016-01-01

    Different types of lipid- and polymer-based vectors have been developed to deliver proteins into cells, but these methods showed relatively poor efficiency. Recently, a group of short, highly basic peptides known as cell-penetrating peptides (CPPs) were used to carry polypeptides and proteins into cells. In this study, expression and purification of GFP protein was performed using the prokaryotic pET expression system. We used two amphipathic CPPs (Pep-1 and CADY-2) as a novel delivery system to transfer the GFP protein into cells. The morphological features of the CPP/GFP complexes were studied by scanning electron microscopy (SEM), Zetasizer, and SDS-PAGE. The efficiency of GFP transfection using Pep-1 and CADY-2 peptides and TurboFect reagent was compared with FITC-antibody protein control delivered by these transfection vehicles in the HEK-293T cell line. SEM data confirmed formation of discrete nanoparticles with a diameter of below 300 nm. Moreover, formation of the complexes was detected using SDS-PAGE as two individual bands, indicating non-covalent interaction. The size and homogeneity of Pep-1/GFP and CADY-2/GFP complexes were dependent on the ratio of peptide/cargo formulations, and responsible for their biological efficiency. The cells transfected by Pep-1/GFP and CADY-2/GFP complexes at a molar ratio of 20 : 1 demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the transfection efficiency of Pep-based nanoparticles was similar to CADY-based nanoparticles and comparable with TurboFect-protein complexes. These data open an efficient way for future therapeutic purposes.

  11. Tritium Suicide Selection Identifies Proteins Involved in the Uptake and Intracellular Transport of Sterols in Saccharomyces cerevisiae▿

    Science.gov (United States)

    Sullivan, David P.; Georgiev, Alexander; Menon, Anant K.

    2009-01-01

    Sterol transport between the plasma membrane (PM) and the endoplasmic reticulum (ER) occurs by a nonvesicular mechanism that is poorly understood. To identify proteins required for this process, we isolated Saccharomyces cerevisiae mutants with defects in sterol transport. We used Upc2-1 cells that have the ability to take up sterols under aerobic conditions and exploited the observation that intracellular accumulation of exogenously supplied [3H]cholesterol in the form of [3H]cholesteryl ester requires an intact PM-ER sterol transport pathway. Upc2-1 cells were mutagenized using a transposon library, incubated with [3H]cholesterol, and subjected to tritium suicide selection to isolate mutants with a decreased ability to accumulate [3H]cholesterol. Many of the mutants had defects in the expression and trafficking of Aus1 and Pdr11, PM-localized ABC transporters that are required for sterol uptake. Through characterization of one of the mutants, a new role was uncovered for the transcription factor Mot3 in controlling expression of Aus1 and Pdr11. A number of mutants had transposon insertions in the uncharacterized Ydr051c gene, which we now refer to as DET1 (decreased ergosterol transport). These mutants expressed Aus1 and Pdr11 normally but were severely defective in the ability to accumulate exogenously supplied cholesterol. The transport of newly synthesized sterols from the ER to the PM was also defective in det1Δ cells. These data indicate that the cytoplasmic protein encoded by DET1 is involved in intracellular sterol transport. PMID:19060182

  12. The TRPC2 channel forms protein-protein interactions with Homer and RTP in the rat vomeronasal organ

    Directory of Open Access Journals (Sweden)

    Brann Jessica H

    2010-05-01

    Full Text Available Abstract Background The signal transduction cascade operational in the vomeronasal organ (VNO of the olfactory system detects odorants important for prey localization, mating, and social recognition. While the protein machinery transducing these external cues has been individually well characterized, little attention has been paid to the role of protein-protein interactions among these molecules. Development of an in vitro expression system for the transient receptor potential 2 channel (TRPC2, which establishes the first electrical signal in the pheromone transduction pathway, led to the discovery of two protein partners that couple with the channel in the native VNO. Results Homer family proteins were expressed in both male and female adult VNO, particularly Homer 1b/c and Homer 3. In addition to this family of scaffolding proteins, the chaperones receptor transporting protein 1 (RTP1 and receptor expression enhancing protein 1 (REEP1 were also expressed. RTP1 was localized broadly across the VNO sensory epithelium, goblet cells, and the soft palate. Both Homer and RTP1 formed protein-protein interactions with TRPC2 in native reciprocal pull-down assays and RTP1 increased surface expression of TRPC2 in in vitro assays. The RTP1-dependent TRPC2 surface expression was paralleled with an increase in ATP-stimulated whole-cell current in an in vitro patch-clamp electrophysiological assay. Conclusions TRPC2 expression and channel activity is regulated by chaperone- and scaffolding-associated proteins, which could modulate the transduction of chemosignals. The developed in vitro expression system, as described here, will be advantageous for detailed investigations into TRPC2 channel activity and cell signalling, for a channel protein that was traditionally difficult to physiologically assess.

  13. The Intracellular Destiny of the Protein Corona : A Study on its Cellular Internalization and Evolution

    NARCIS (Netherlands)

    Bertoli, Filippo; Garry, David; Monopoli, Marco P.; Salvati, Anna; Dawson, Kenneth A.

    2016-01-01

    It has been well established that the early stages of nanoparticle cell interactions are governed, at least in part, by the layer of proteins and other biomolecules adsorbed and slowly exchanged with the surrounding biological media (biomolecular corona). Subsequent to membrane interactions,

  14. Photo-oxidation of cells generates long-lived intracellular protein peroxides

    DEFF Research Database (Denmark)

    Wright, Adam; Hawkins, Clare Louise; Davies, Michael Jonathan

    2003-01-01

    Singlet oxygen is generated by several cellular, enzymatic, and chemical reactions as well as by exposure to UV or visible light in the presence of a sensitizer. Consequently, this oxidant has been proposed to be a damaging agent many pathologies. Proteins are major targets for singlet oxygen as ...

  15. Intracellular localization of rice stripe virus RNA-dependent RNA polymerase and its interaction with nucleocapsid protein.

    Science.gov (United States)

    Zhao, Shuling; Hao, Jiahui; Xue, Yanan; Liang, Changyong

    2015-12-01

    The RNA-dependent RNA polymerase (RdRp) of rice stripe virus (RSV) is critical for both the transcription and replication of the viral genome. Despite its importance, little is known about how it functions in cells. In the present study, RSV RdRp was split into three pieces, since expression of the full protein could not be achieved. Then, the intracellular localization of these three RdRp fragments and their interactions with nucleocapsid protein (NP) were investigated, which is another viral protein required for viral RNA synthesis. The data showed that all three RdRp fragments displayed punctuate staining patterns in the cytoplasm, and the C-terminal fragment co-localized with NP in the perinuclear region. Both bimolecular fluorescence complementation and co-immunoprecipitation experiments demonstrated that of the three RdRp fragments, only the C-terminal fragment could interact with NP. Further analysis using a series of truncated NPs identified the N-terminal 50-amino-acid region within NP as the determinant for its interaction with the C-terminus of RdRp.

  16. Loss of Selenium-Binding Protein 1 Decreases Sensitivity to Clastogens and Intracellular Selenium Content in HeLa Cells.

    Science.gov (United States)

    Zhao, Changhui; Zeng, Huawei; Wu, Ryan T Y; Cheng, Wen-Hsing

    2016-01-01

    Selenium-binding protein 1 (SBP1) is not a selenoprotein but structurally binds selenium. Loss of SBP1 during carcinogenesis usually predicts poor prognosis. Because genome instability is a hallmark of cancer, we hypothesize that SBP1 sequesters cellular selenium and sensitizes cancer cells to DNA-damaging agents. To test this hypothesis, we knocked down SBP1 expression in HeLa cervical cancer cells by employing a short hairpin RNA (shRNA) approach. Reduced sensitivity to hydrogen peroxide, paraquat and camptothecin, reactive oxygen species content, and intracellular retention of selenium after selenomethionine treatment were observed in SBP1 shRNA HeLa cells. Results from Western analyses showed that treatment of HeLa cells with selenomethionine resulted in increased SBP1 protein expression in a dose-dependent manner. Knockdown of SBP1 rendered HeLa cells increased expression of glutathione peroxidase-1 but not glutathione peroxidase-4 protein levels and accelerated migration from a wound. Altogether, SBP1 retains supplemental selenium and sensitizes HeLa cancer cells to clastogens, suggesting a new cancer treatment strategy by sequestering selenium through SBP1.

  17. Conserved Ankyrin Repeat Proteins and Their NIMA Kinase Partners Regulate Extracellular Matrix Remodeling and Intracellular Trafficking in Caenorhabditis elegans.

    Science.gov (United States)

    Lažetić, Vladimir; Fay, David S

    2017-01-01

    Molting is an essential developmental process in nematodes during which the epidermal apical extracellular matrix, the cuticle, is remodeled to accommodate further growth. Using genetic approaches, we identified a requirement for three conserved ankyrin repeat-rich proteins, MLT-2/ANKS6, MLT-3/ANKS3, and MLT-4/INVS, in Caenorhabditis elegans molting. Loss of mlt function resulted in severe defects in the ability of larvae to shed old cuticle and led to developmental arrest. Genetic analyses demonstrated that MLT proteins functionally cooperate with the conserved NIMA kinase family members NEKL-2/NEK8 and NEKL-3/NEK6/NEK7 to promote cuticle shedding. MLT and NEKL proteins were specifically required within the hyp7 epidermal syncytium, and fluorescently tagged mlt and nekl alleles were expressed in puncta within this tissue. Expression studies further showed that NEKL-2-MLT-2-MLT-4 and NEKL-3-MLT-3 colocalize within largely distinct assemblies of apical foci. MLT-2 and MLT-4 were required for the normal accumulation of NEKL-2 at the hyp7-seam cell boundary, and loss of mlt-2 caused abnormal nuclear accumulation of NEKL-2 Correspondingly, MLT-3, which bound directly to NEKL-3, prevented NEKL-3 nuclear localization, supporting the model that MLT proteins may serve as molecular scaffolds for NEKL kinases. Our studies additionally showed that the NEKL-MLT network regulates early steps in clathrin-mediated endocytosis at the apical surface of hyp7, which may in part account for molting defects observed in nekl and mlt mutants. This study has thus identified a conserved NEKL-MLT protein network that regulates remodeling of the apical extracellular matrix and intracellular trafficking, functions that may be conserved across species. Copyright © 2017 by the Genetics Society of America.

  18. Effects of inhibitors of protein synthesis and intracellular transport on the gamma-aminobutyric acid agonist-induced functional differentiation of cultured cerebellar granule cells

    DEFF Research Database (Denmark)

    Belhage, B; Hansen, Gert Helge; Meier, E

    1990-01-01

    The effect of inhibitors of protein synthesis (actinomycin D, cycloheximide), proteases (leupeptin), and intracellular transport (colchicine, monensin) on the gamma-aminobutyric acid (GABA) agonist [4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP)]-induced changes in morphological differenti......The effect of inhibitors of protein synthesis (actinomycin D, cycloheximide), proteases (leupeptin), and intracellular transport (colchicine, monensin) on the gamma-aminobutyric acid (GABA) agonist [4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP)]-induced changes in morphological...

  19. A docking study of enhanced intracellular survival protein from Mycobacterium tuberculosis with human DUSP16/MKP-7

    International Nuclear Information System (INIS)

    Yoon, Hye-Jin; Kim, Kyoung Hoon; Yang, Jin Kuk; Suh, Se Won; Kim, Hyunsik; Jang, Soonmin

    2013-01-01

    A docking study of Mtb Eis with its substrate DUSP16/MKP-7 was performed. The docking model suggests dissociation of hexameric Mtb Eis into dimers or monomers. The intracellular pathogen Mycobacterium tuberculosis (Mtb) causes tuberculosis, and one of its secreted effector proteins, called enhanced intracellular survival (Eis) protein, enhances its survival in macrophages. Mtb Eis activates JNK-specific dual-specificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase-7 (MKP-7) through the acetylation on Lys55, thus inactivating JNK by dephosphorylation. Based on the recently reported crystal structure of Mtb Eis, a docking model for the binding of Mtb Eis to DUSP16/MKP-7 was generated. In the docking model, the substrate helix containing Lys55 of DUSP16/MKP-7 fits nicely into the active-site cleft of Mtb Eis; the twisted β-sheet of Eis domain II embraces the substrate helix from one side. Most importantly, the side-chain of Lys55 is inserted toward acetyl-CoA and the resulting distance is 4.6 Å between the NZ atom of Lys55 and the carbonyl carbon of the acetyl group in acetyl-CoA. The binding of Mtb Eis and DUSP16/MKP-7 is maintained by strong electrostatic interactions. The active-site cleft of Mtb Eis has a negatively charged surface formed by Asp25, Glu138, Asp286, Glu395 and the terminal carboxylic group of Phe396. In contrast, DUSP16/MKP-7 contains five basic residues, Lys52, Lys55, Arg56, Arg57 and Lys62, which point toward the negatively charged surface of the active-site pocket of Mtb Eis. Thus, the current docking model suggests that the binding of DUSP16/MKP-7 to Mtb Eis should be established by charge complementarity in addition to a very favorable geometric arrangement. The suggested mode of binding requires the dissociation of the hexameric Mtb Eis into dimers or monomers. This study may be useful for future studies aiming to develop inhibitors of Mtb Eis as a new anti-tuberculosis drug candidate

  20. Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5

    International Nuclear Information System (INIS)

    Goda, Natsuko; Tenno, Takeshi; Inomata, Kosuke; Shirakawa, Masahiro; Tanaka, Toshiki; Hiroaki, Hidekazu

    2008-01-01

    Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs

  1. Fluoride export (FEX proteins from fungi, plants and animals are 'single barreled' channels containing one functional and one vestigial ion pore.

    Directory of Open Access Journals (Sweden)

    Tetyana Berbasova

    Full Text Available The fluoride export protein (FEX in yeast and other fungi provides tolerance to fluoride (F-, an environmentally ubiquitous anion. FEX efficiently eliminates intracellular fluoride that otherwise would accumulate at toxic concentrations. The FEX homolog in bacteria, Fluc, is a 'double-barreled' channel formed by dimerization of two identical or similar subunits. FEX in yeast and other eukaryotes is a monomer resulting from covalent fusion of the two subunits. As a result, both potential fluoride pores are created from different parts of the same protein. Here we identify FEX proteins from two multicellular eukaryotes, a plant Arabidopsis thaliana and an animal Amphimedon queenslandica, by demonstrating significant fluoride tolerance when these proteins are heterologously expressed in the yeast Saccharomyces cerevisiae. Residues important for eukaryotic FEX function were determined by phylogenetic sequence alignment and functional analysis using a yeast growth assay. Key residues of the fluoride channel are conserved in only one of the two potential fluoride-transporting pores. FEX activity is abolished upon mutation of residues in this conserved pore, suggesting that only one of the pores is functional. The same topology is conserved for the newly identified FEX proteins from plant and animal. These data suggest that FEX family of fluoride channels in eukaryotes are 'single-barreled' transporters containing one functional pore and a second non-functional vestigial remnant of a homologous gene fusion event.

  2. Fragile X mental retardation protein controls ion channel expression and activity.

    Science.gov (United States)

    Ferron, Laurent

    2016-10-15

    Fragile X-associated disorders are a family of genetic conditions resulting from the partial or complete loss of fragile X mental retardation protein (FMRP). Among these disorders is fragile X syndrome, the most common cause of inherited intellectual disability and autism. FMRP is an RNA-binding protein involved in the control of local translation, which has pleiotropic effects, in particular on synaptic function. Analysis of the brain FMRP transcriptome has revealed hundreds of potential mRNA targets encoding postsynaptic and presynaptic proteins, including a number of ion channels. FMRP has been confirmed to bind voltage-gated potassium channels (K v 3.1 and K v 4.2) mRNAs and regulates their expression in somatodendritic compartments of neurons. Recent studies have uncovered a number of additional roles for FMRP besides RNA regulation. FMRP was shown to directly interact with, and modulate, a number of ion channel complexes. The sodium-activated potassium (Slack) channel was the first ion channel shown to directly interact with FMRP; this interaction alters the single-channel properties of the Slack channel. FMRP was also shown to interact with the auxiliary β4 subunit of the calcium-activated potassium (BK) channel; this interaction increases calcium-dependent activation of the BK channel. More recently, FMRP was shown to directly interact with the voltage-gated calcium channel, Ca v 2.2, and reduce its trafficking to the plasma membrane. Studies performed on animal models of fragile X syndrome have revealed links between modifications of ion channel activity and changes in neuronal excitability, suggesting that these modifications could contribute to the phenotypes observed in patients with fragile X-associated disorders. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

  3. Gene expression and population polymorphism of maize Iranian mosaic virus in Zea mays, and intracellular localization and interactions of viral N, P, and M proteins in Nicotiana benthamiana.

    Science.gov (United States)

    Ghorbani, Abozar; Izadpanah, Keramatollah; Dietzgen, Ralf G

    2018-02-15

    Maize Iranian mosaic virus (MIMV; Mononegavirales, Rhabdoviridae, Nucleorhabdovirus) infects maize and several other poaceous plants. MIMV encodes six proteins, i.e., nucleocapsid protein (N), polymerase cofactor phosphoprotein (P), putative movement protein (P3), matrix protein (M), glycoprotein (G), and large RNA-dependent RNA polymerase (L). In the present study, MIMV gene expression and genetic polymorphism of an MIMV population in maize were determined. N, P, P3, and M protein genes were more highly expressed than the 5' terminal G and L genes. Twelve single nucleotide polymorphisms were identified across the genome within a MIMV population in maize from RNA-Seq read data pooled from three infected plants indicating genomic variations of potential importance to evolution of the virus. MIMV N, P, and M proteins that are known to be involved in rhabdovirus replication and transcription were characterized as to their intracellular localization and interactions. N protein accumulated exclusively in the nucleus and interacted with itself and with P protein. P protein accumulated in both the nucleus and cell periphery and interacted with itself, N and M proteins in the nucleus. M protein was localized in the cell periphery and on endomembranes, and interacted with P protein in the nucleus. MIMV proteins show a distinctive combination of intracellular localizations and interactions.

  4. Inhibition of TREK-2 K(+) channels by PI(4,5)P2: an intrinsic mode of regulation by intracellular ATP via phosphatidylinositol kinase.

    Science.gov (United States)

    Woo, Joohan; Shin, Dong Hoon; Kim, Hyun Jong; Yoo, Hae Young; Zhang, Yin-Hua; Nam, Joo Hyun; Kim, Woo Kyung; Kim, Sung Joon

    2016-08-01

    TWIK-related two-pore domain K(+) channels 1 and 2 (TREKs) are activated under various physicochemical conditions. However, the directions in which they are regulated by PI(4,5)P2 and intracellular ATP are not clearly presented yet. In this study, we investigated the effects of ATP and PI(4,5)P2 on overexpressed TREKs (HEK293T and COS-7) and endogenously expressed TREK-2 (mouse astrocytes and WEHI-231 B cells). In all of these cells, both TREK-1 and TREK-2 currents were spontaneously increased by dialysis with ATP-free pipette solution for whole-cell recording (ITREK-1,w-c and ITREK-2w-c) or by membrane excision for inside-out patch clamping without ATP (ITREK-1,i-o and ITREK-2,i-o). Steady state ITREK-2,i-o was reversibly decreased by 3 mM ATP applied to the cytoplasmic side, and this reduction was prevented by wortmannin, a PI-kinase inhibitor. An exogenous application of PI(4,5)P2 inhibited the spontaneously increased ITREKs,i-o, suggesting that intrinsic PI(4,5)P2 maintained by intracellular ATP and PI kinase may set the basal activity of TREKs in the intact cells. The inhibition of intrinsic TREK-2 by ATP was more prominent in WEHI-231 cells than astrocytes. Interestingly, unspecific screening of negative charges by poly-L-lysine also inhibited ITREK-2,i-o. Application of PI(4,5)P2 after the poly-L-lysine treatment showed dose-dependent dual effects, initial activation and subsequent inhibition of ITREK-2,i-o at low and high concentrations, respectively. In HEK293T cells coexpressing TREK-2 and a voltage-sensitive PI(4,5)P2 phosphatase, sustained depolarization increased ITREK-2,w-c initially (PI(4,5)P2 suggests the existence of dual regulatory modes that depend on PI(4,5)P2 concentration.

  5. Intracellular localization of Xenopus small heat shock protein, hsp30, in A6 kidney epithelial cells.

    Science.gov (United States)

    Gellalchew, Mekonnen; Heikkila, John J

    2005-03-01

    Small heat shock proteins (shsps) are molecular chaperones that are inducible by environmental stress. In this study, immunocytochemical analysis and laser scanning confocal microscopy revealed that the shsp family, hsp30, was localized primarily in the cytoplasm of Xenopus A6 kidney epithelial cells after heat shock or sodium arsenite treatment. Heat shock-induced hsp30 was enriched in the perinuclear region with some immunostaining in the nucleus but not in the nucleolus. In sodium arsenite-treated cells hsp30 was enriched towards the cytoplasmic periphery as well as showing some immunostaining in the nucleus. At higher heat shock temperatures (35 degrees C) or after 10 microM sodium arsenite treatment, the actin cytoskeleton displayed some disorganization that co-localized with areas of hsp30 enrichment. Treatment of A6 cells with 50 microM sodium arsenite induced a collapse of the cytoskeleton around the nucleus. These results coupled with previous studies suggest that stress-inducible hsp30 acts as a molecular chaperone primarily in the cytoplasm and may interact with cytoskeletal proteins.

  6. Isonicotinamide Enhances Sir2 Protein-mediated Silencing and Longevity in Yeast by Raising Intracellular NAD+ Concentration*

    Science.gov (United States)

    McClure, Julie M.; Wierman, Margaret B.; Maqani, Nazif; Smith, Jeffrey S.

    2012-01-01

    Sirtuins are an evolutionarily conserved family of NAD+-dependent protein deacetylases that function in the regulation of gene transcription, cellular metabolism, and aging. Their activity requires the maintenance of an adequate intracellular NAD+ concentration through the combined action of NAD+ biosynthesis and salvage pathways. Nicotinamide (NAM) is a key NAD+ precursor that is also a byproduct and feedback inhibitor of the deacetylation reaction. In Saccharomyces cerevisiae, the nicotinamidase Pnc1 converts NAM to nicotinic acid (NA), which is then used as a substrate by the NAD+ salvage pathway enzyme NA phosphoribosyltransferase (Npt1). Isonicotinamide (INAM) is an isostere of NAM that stimulates yeast Sir2 deacetylase activity in vitro by alleviating the NAM inhibition. In this study, we determined that INAM stimulates Sir2 through an additional mechanism in vivo, which involves elevation of the intracellular NAD+ concentration. INAM enhanced normal silencing at the rDNA locus but only partially suppressed the silencing defects of an npt1Δ mutant. Yeast cells grown in media lacking NA had a short replicative life span, which was extended by INAM in a SIR2-dependent manner and correlated with increased NAD+. The INAM-induced increase in NAD+ was strongly dependent on Pnc1 and Npt1, suggesting that INAM increases flux through the NAD+ salvage pathway. Part of this effect was mediated by the NR salvage pathways, which generate NAM as a product and require Pnc1 to produce NAD+. We also provide evidence suggesting that INAM influences the expression of multiple NAD+ biosynthesis and salvage pathways to promote homeostasis during stationary phase. PMID:22539348

  7. Isonicotinamide enhances Sir2 protein-mediated silencing and longevity in yeast by raising intracellular NAD+ concentration.

    Science.gov (United States)

    McClure, Julie M; Wierman, Margaret B; Maqani, Nazif; Smith, Jeffrey S

    2012-06-15

    Sirtuins are an evolutionarily conserved family of NAD(+)-dependent protein deacetylases that function in the regulation of gene transcription, cellular metabolism, and aging. Their activity requires the maintenance of an adequate intracellular NAD(+) concentration through the combined action of NAD(+) biosynthesis and salvage pathways. Nicotinamide (NAM) is a key NAD(+) precursor that is also a byproduct and feedback inhibitor of the deacetylation reaction. In Saccharomyces cerevisiae, the nicotinamidase Pnc1 converts NAM to nicotinic acid (NA), which is then used as a substrate by the NAD(+) salvage pathway enzyme NA phosphoribosyltransferase (Npt1). Isonicotinamide (INAM) is an isostere of NAM that stimulates yeast Sir2 deacetylase activity in vitro by alleviating the NAM inhibition. In this study, we determined that INAM stimulates Sir2 through an additional mechanism in vivo, which involves elevation of the intracellular NAD(+) concentration. INAM enhanced normal silencing at the rDNA locus but only partially suppressed the silencing defects of an npt1Δ mutant. Yeast cells grown in media lacking NA had a short replicative life span, which was extended by INAM in a SIR2-dependent manner and correlated with increased NAD(+). The INAM-induced increase in NAD(+) was strongly dependent on Pnc1 and Npt1, suggesting that INAM increases flux through the NAD(+) salvage pathway. Part of this effect was mediated by the NR salvage pathways, which generate NAM as a product and require Pnc1 to produce NAD(+). We also provide evidence suggesting that INAM influences the expression of multiple NAD(+) biosynthesis and salvage pathways to promote homeostasis during stationary phase.

  8. Common pharmacophore of structurally distinct small-molecule inhibitors of intracellular retrograde trafficking of ribosome inactivating proteins.

    Science.gov (United States)

    Yu, Shichao; Park, Jewn Giew; Kahn, Jennifer Nielsen; Tumer, Nilgun E; Pang, Yuan-Ping

    2013-12-02

    We reported previously (±)-2-(5-methylthiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one [(±)-Retro-2(cycl)] as the chemical structure of Retro-2 that showed mouse protection against ricin, a notorious ribosome inactivating protein (RIP). Herein we report our chemical resolution of (±)-Retro-2(cycl), analog synthesis, and cell-based evaluation showing that the two optically pure enantiomers and their achiral analog have nearly the same degree of cell protection against ricin as (±)-Retro-2(cycl). We also report our computational studies explaining the lack of stereo preference and revealing a common pharmacophore of structurally distinct inhibitors of intracellular retrograde trafficking of RIPs. This pharmacophore comprises a central aromatic ring o-substituted by an aromatic ring and a moiety bearing an O or S atom attached to sp² C atom(s). These results offer new insights into lead identification and optimization for RIP antidote development to minimize the global health threat caused by ribosome-inactivating proteins.

  9. Different intracellular distribution of avian reovirus core protein sigmaA in cells of avian and mammalian origin

    Energy Technology Data Exchange (ETDEWEB)

    Vazquez-Iglesias, Lorena; Lostale-Seijo, Irene; Martinez-Costas, Jose [Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, y Centro Singular de Investigacion en Quimica Biologica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782-Santiago de Compostela (Spain); Benavente, Javier, E-mail: franciscojavier.benavente@usc.es [Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, y Centro Singular de Investigacion en Quimica Biologica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782-Santiago de Compostela (Spain)

    2012-10-25

    A comparative analysis of the intracellular distribution of avian reovirus (ARV) core protein sigmaA in cells of avian and mammalian origin revealed that, whereas the viral protein accumulates in the cytoplasm and nucleolus of avian cells, most sigmaA concentrates in the nucleoplasm of mammalian cells in tight association with the insoluble nuclear matrix fraction. Our results further showed that sigmaA becomes arrested in the nucleoplasm of mammalian cells via association with mammalian cell-specific factors and that this association prevents nucleolar targeting. Inhibition of RNA polymerase II activity, but not of RNA polymerase I activity, in infected mammalian cells induces nucleus-to-cytoplasm sigmaA translocation through a CRM1- and RanGTP-dependent mechanism, yet a heterokaryon assay suggests that sigmaA does not shuttle between the nucleus and cytoplasm. The scarcity of sigmaA in cytoplasmic viral factories of infected mammalian cells could be one of the factors contributing to limited ARV replication in mammalian cells.

  10. Effects of inhibitors of protein synthesis and intracellular transport on the gamma-aminobutyric acid agonist-induced functional differentiation of cultured cerebellar granule cells

    DEFF Research Database (Denmark)

    Belhage, B; Hansen, Gert Helge; Meier, E

    1990-01-01

    an intracellular and a plasma membrane localization of the receptors. In all experiments cultures treated with THIP alone served as controls. The inhibitors of protein synthesis totally abolished the ability of THIP to induce low-affinity GABA receptors. In contrast, the inhibitors of intracellular transport......The effect of inhibitors of protein synthesis (actinomycin D, cycloheximide), proteases (leupeptin), and intracellular transport (colchicine, monensin) on the gamma-aminobutyric acid (GABA) agonist [4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP)]-induced changes in morphological...... as well as the protease inhibitor did not affect this parameter. However, studies of effects of GABA on transmitter release from monensin-treated cultures showed that transmitter release could not be inhibited by GABA in these cells in spite of the presence of low-affinity GABA receptors in the membrane...

  11. Angiotensin-2-mediated Ca2+ signaling in the retinal pigment epithelium: role of angiotensin-receptor-associated-protein and TRPV2 channel.

    Directory of Open Access Journals (Sweden)

    Rene Barro-Soria

    Full Text Available Angiotensin II (AngII receptor (ATR is involved in pathologic local events such as neovascularisation and inflammation including in the brain and retina. The retinal pigment epithelium (RPE expresses ATR in its AT1R form, angiotensin-receptor-associated protein (Atrap, and transient-receptor-potential channel-V2 (TRPV2. AT1R and Atrap co-localize to the basolateral membrane of the RPE, as shown by immunostaining. Stimulation of porcine RPE (pRPE cells by AngII results in biphasic increases in intracellular free Ca(2+inhibited by losartan. Xestospongin C (xest C and U-73122, blockers of IP3R and PLC respectively, reduced AngII-evoked Ca(2+response. RPE cells from Atrap(-/- mice showed smaller AngII-evoked Ca(2+peak (by 22% and loss of sustained Ca(2+elevation compared to wild-type. The TRPV channel activator cannabidiol (CBD at 15 µM stimulates intracellular Ca(2+-rise suggesting that porcine RPE cells express TRPV2 channels. Further evidence supporting the functional expression of TRPV2 channels comes from experiments in which 100 µM SKF96365 (a TRPV channel inhibitor reduced the cannabidiol-induced Ca(2+-rise. Application of SKF96365 or reduction of TRPV2 expression by siRNA reduced the sustained phase of AngII-mediated Ca(2+transients by 53%. Thus systemic AngII, an effector of the local renin-angiotensin system stimulates biphasic Ca(2+transients in the RPE by releasing Ca(2+from cytosolic IP3-dependent stores and activating ATR/Atrap and TRPV2 channels to generate a sustained Ca(2+elevation.

  12. Nuclear localization of DMP1 proteins suggests a role in intracellular signaling

    Energy Technology Data Exchange (ETDEWEB)

    Siyam, Arwa [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 (United States); Wang, Suzhen; Qin, Chunlin; Mues, Gabriele [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Stevens, Roy [Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 (United States); D' Souza, Rena N. [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Lu, Yongbo, E-mail: ylu@bcd.tamhsc.edu [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Nuclear localization of DMP1 in various cell lines. Black-Right-Pointing-Pointer Non-synchronized cells show either nuclear or cytoplasmic localization of DMP1. Black-Right-Pointing-Pointer Nuclear DMP1 is restricted to the nucleoplasm but absent in the nucleolus. -- Abstract: Dentin matrix protein 1 (DMP1) is highly expressed in odontoblasts and osteoblasts/osteocytes and plays an essential role in tooth and bone mineralization and phosphate homeostasis. It is debatable whether DMP1, in addition to its function in the extracellular matrix, can enter the nucleus and function as a transcription factor. To better understand its function, we examined the nuclear localization of endogenous and exogenous DMP1 in C3H10T1/2 mesenchymal cells, MC3T3-E1 preosteoblast cells and 17IIA11 odontoblast-like cells. RT-PCR analyses showed the expression of endogenous Dmp1 in all three cell lines, while Western-blot analysis detected a major DMP1 protein band corresponding to the 57 kDa C-terminal fragment generated by proteolytic processing of the secreted full-length DMP1. Immunofluorescent staining demonstrated that non-synchronized cells presented two subpopulations with either nuclear or cytoplasmic localization of endogenous DMP1. In addition, cells transfected with a construct expressing HA-tagged full-length DMP1 also showed either nuclear or cytoplasmic localization of the exogenous DMP1 when examined with an antibody against the HA tag. Furthermore, nuclear DMP1 was restricted to the nucleoplasm but was absent in the nucleolus. In conclusion, these findings suggest that, apart from its role as a constituent of dentin and bone matrix, DMP1 might play a regulatory role in the nucleus.

  13. Macromolecular crowding enhances the binding of superoxide dismutase to xanthine oxidase: implications for protein-protein interactions in intracellular environments.

    Science.gov (United States)

    Zhou, Yu-Ling; Liao, Jun-Ming; Chen, Jie; Liang, Yi

    2006-01-01

    Physiological medium constitutes a crowded environment that serves as the field of action for protein-protein interaction in vivo. Measuring protein-protein interaction in crowded solutions can mimic this environment. Here we report the application of fluorescence spectroscopy and resonant mirror biosensor to investigate the interactions of bovine milk xanthine oxidase and bovine erythrocyte copper, zinc-superoxide dismutase in crowded solutions. Four nonspecific high molecular mass crowding agents, poly(ethylene glycol) 2000 and 20,000, Ficoll 70, and dextran 70, and one low molecular mass compound, glycerol, are used. Superoxide dismutase shows a strong and macromolecular crowding agent concentration-dependent binding affinity to xanthine oxidase. Addition of high concentrations of such high molecular mass crowding agents increases the binding constant remarkably and thus stabilizes superoxide dismutase activity, compared to those in the absence of crowding agents. In contrast, glycerol has little effect on the binding constant and decreases superoxide dismutase activity over the same concentration range. Such a pattern suggests that the enhancing effects of polymers and polysaccharides on the binding are due to macromolecular crowding. Taken together, these results indicate that macromolecular crowding enhances the binding of superoxide dismutase to xanthine oxidase and is favorable to the function of superoxide dismutase.

  14. Side-effects of protein kinase inhibitors on ion channels

    Indian Academy of Sciences (India)

    2013-11-06

    Nov 6, 2013 ... In fact, the side-effects have precluded these inhibitors from being both useful experimental tools and therapeutically viable. ... protein kinases both for experimental tools and for therapeutic approaches. [Son YK, Park H, Firth AL ...... of MAPK-activated proteins by MAPK controls the expression of various ...

  15. Side-effects of protein kinase inhibitors on ion channels

    Indian Academy of Sciences (India)

    Protein kinases are one of the largest gene families and have regulatory roles in all aspects of eukaryotic cell function. Modulation of protein kinase activity is a desirable therapeutic approach for a number of human diseases associated with aberrant kinase activity, including cancers, arthritis and cardiovascular disorders.

  16. Structure of synaptophysin: a hexameric MARVEL-domain channel protein.

    Science.gov (United States)

    Arthur, Christopher P; Stowell, Michael H B

    2007-06-01

    Synaptophysin I (SypI) is an archetypal member of the MARVEL-domain family of integral membrane proteins and one of the first synaptic vesicle proteins to be identified and cloned. Most all MARVEL-domain proteins are involved in membrane apposition and vesicle-trafficking events, but their precise role in these processes is unclear. We have purified mammalian SypI and determined its three-dimensional (3D) structure by using electron microscopy and single-particle 3D reconstruction. The hexameric structure resembles an open basket with a large pore and tenuous interactions within the cytosolic domain. The structure suggests a model for Synaptophysin's role in fusion and recycling that is regulated by known interactions with the SNARE machinery. This 3D structure of a MARVEL-domain protein provides a structural foundation for understanding the role of these important proteins in a variety of biological processes.

  17. Differential effects of two-pore channel protein 1 and 2 silencing in MDA-MB-468 breast cancer cells.

    Science.gov (United States)

    Jahidin, Aisyah H; Stewart, Teneale A; Thompson, Erik W; Roberts-Thomson, Sarah J; Monteith, Gregory R

    2016-09-02

    Two-pore channel proteins, TPC1 and TPC2, are calcium permeable ion channels found localized to the membranes of endolysosomal calcium stores. There is increasing interest in the role of TPC-mediated intracellular signaling in various pathologies; however their role in breast cancer has not been extensively evaluated. TPC1 and TPC2 mRNA was present in all non-tumorigenic and tumorigenic breast cell lines assessed. Silencing of TPC2 but not TPC1 attenuated epidermal growth factor-induced vimentin expression in MDA-MB-468 breast cancer cells. This effect was not due to a general inhibition of epithelial to mesenchymal transition (EMT) as TPC2 silencing had no effect on epidermal growth factor (EGF)-induced changes on E-cadherin expression. TPC1 and TPC2 were also shown to differentially regulate cyclopiazonic acid (CPA)-mediated changes in cytosolic free Ca(2+). These findings indicate potential differential regulation of signaling processes by TPC1 and TPC2 in breast cancer cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Evaluation of Peanut Meal as an Alternative Dietary Protein Source for Channel Catfish

    Science.gov (United States)

    Use of peanut meal as an alternative protein source in diets for channel catfish Ictalurus punctatus was evaluated in a 9-week study under controlled laboratory conditions. Five practical diets (28% crude protein and 6% crude lipid) were formulated to contain 0, 10, 15, 20, and 25% peanut meal as a ...

  19. The B7-1 cytoplasmic tail enhances intracellular transport and mammalian cell surface display of chimeric proteins in the absence of a linear ER export motif.

    Directory of Open Access Journals (Sweden)

    Yi-Chieh Lin

    Full Text Available Membrane-tethered proteins (mammalian surface display are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.

  20. Primary structures of two ribonucleases from ginseng calluses - New members of the PR-10 family of intracellular pathogenesis-related plant proteins

    NARCIS (Netherlands)

    Moiseyev, GP; Fedoreyeva, LI; Zhuravlev, YN; Yasnetskaya, E; Jekel, PA; Beintema, JJ

    1997-01-01

    The amino acid sequences of two ribonucleases from a callus cell culture of Panax ginseng were determined, The two sequences differ at 26% of the amino acid positions, Homology was found with a large family of intracellular pathogenesis-related proteins, food allergens and tree pollen allergens from

  1. Mammalian farnesyltransferase α subunit regulates vacuolar protein sorting-associated protein 4A (Vps4A)--dependent intracellular trafficking through recycling endosomes.

    Science.gov (United States)

    Kubala, Marta H; Norwood, Suzanne J; Gomez, Guillermo A; Jones, Alun; Johnston, Wayne; Yap, Alpha S; Mureev, Sergey; Alexandrov, Kirill

    2015-12-25

    The protein farnesyltransferase (FTase) mediates posttranslational modification of proteins with isoprenoid lipids. FTase is a heterodimer and although the β subunit harbors the active site, it requires the α subunit for its activity. Here we explore the other functions of the FTase α subunit in addition to its established role in protein prenylation. We found that in the absence of the β subunit, the α subunit of FTase forms a stable autonomous dimeric structure in solution. We identify interactors of FTase α using mass spectrometry, followed by rapid in vitro analysis using the Leishmania tarentolae cell - free system. Vps4A was validated for direct binding to the FTase α subunit both in vitro and in vivo. Analysis of the interaction with Vps4A in Hek 293 cells demonstrated that FTase α controls trafficking of transferrin receptor upstream of this protein. These results point to the existence of previously undetected biological functions of the FTase α subunit that includes control of intracellular membrane trafficking. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Discovery of novel DENN proteins: implications for the evolution of eukaryotic intracellular membrane structures and human disease

    Directory of Open Access Journals (Sweden)

    Dapeng eZhang

    2012-12-01

    Full Text Available The tripartite DENN module, comprised of a N-terminal longin domain, followed by DENN and d-DENN domains, is a GDP-GTP exchange factor (GEFs for Rab GTPases, which are regulators of practically all membrane trafficking events in eukaryotes. Using sequence and structure analysis we identify multiple novel homologs of the DENN module, many of which can be traced back to the ancestral eukaryote. These findings provide unexpected leads regarding key cellular processes such as autophagy, vesicle-vacuole interactions, chromosome segregation and human disease. Of these, SMCR8, the folliculin interacting protein-1 and 2 (FNIP1 and FNIP2, nitrogen permease regulator 2 (NPR2 and NPR3 are proposed to function in recruiting Rab GTPases during different steps of autophagy, fusion of autophagosomes with the vacuole and regulation of cellular metabolism. Another novel DENN protein identified in this study is C9ORF72; expansions of the hexanucleotide GGGGCC in its first intron have been recently implicated in amyotrophic lateral sclerosis (ALS and fronto-temporal dementia (FTD. While this mutation is proposed to cause a RNA-level defect, the identification of C9ORF72 as a potential DENN-type GEF raises the possibility that at least part of the pathology might relate to a specific Rab-dependent vesicular trafficking process, as has been observed in the case of some other neurological conditions with similar phenotypes. We present evidence that the longin domain, such as those found in the DENN module, are likely to have been ultimately derived from the related domains found in prokaryotic GTPase-activating proteins of MglA-like GTPases. Thus, the origin of the longin domains from this ancient GTPase-interacting domain, concomitant with the radiation of GTPases, especially of the Rab clade, played an important role in the dynamics of eukaryotic intracellular membrane systems.

  3. LC/MS identification of 12 intracellular cytoskeletal and inflammatory proteins from monocytes adherent on surface-adsorbed fibronectin-derived peptides

    OpenAIRE

    Zuckerman, Sean T.; Kao, Weiyuan John

    2008-01-01

    The extent and duration of the host response determines device efficacy yet the mechanism is poorly understood. U937 pro-monocytic cells were cultured on peptide-adsorbed tissue culture polystyrene to better understand surface-modulated intracellular events. Phosphotyrosine proteins were enriched by immunoprecipitation and analyzed by nanospray HPLC-coupled tandem mass spectrometry (LC/MS). Tyrosine phosphorylated proteins were chosen based on physiological significance and previous densitome...

  4. Intracellular co-localization of SPLUNC1 protein with nanobacteria in nasopharyngeal carcinoma epithelia HNE1 cells depended on the bactericidal permeability increasing protein domain.

    Science.gov (United States)

    Zhou, Hou-De; Li, Gui-Yuan; Yang, Yi-Xin; Li, Xiao-Ling; Sheng, Shou-Rong; Zhang, Wen-Ling; Zhao, Jin

    2006-04-01

    Epithelial surfaces constitute natural immunobarriers against environmental threats. These barriers are brimming with fluids that bind, transport, cleave or degrade bacterial cells and their endotoxic by-products. Saliva and the airway surface-lining fluid (ASL) comprise the important fluid constituents. Short palate, lung and nasal epithelium clone 1 (SPLUNC1) is a potential host defensive protein that is secreted from the submucosal gland to the saliva and nasal lavage fluid. However, its antimicrobial spectrum and antimicrobial mechanism is not clear. Through green fluorescence protein (GFP) mediated subcellular localization experiments in nasopharyngeal carcinoma (NPC) HNE1 cell line, we determined that the intracellular GFP-tagged SPLUNC1 protein binds to a miniscule microorganisms, approximately 50-400nm in size, after the bactericidal permeability increasing protein (BPI) domain was deleted, GFP-tagged truncated SPLUNC1 protein lost its function of binding to the miniscule microorganisms. We verified that these microorganisms are nanobacteria (NB) with a negative staining using transmitted electronic microscope (TEM) and immunofluorescent analysis using an NB-specific antibody. We isolated and cultured the NB from the cultured nasopharyngeal carcinoma epithelia HNE1 cell supernatant. We found that the NB did not absorb the Hoechst stain, even when we extended the staining time to 35min. However, with the time extension the larger sized NB (larger than 300nm) did stain positively. From the biopsy specimen of NPC, we also detected the NB, which can lead to the swelling of mitochondria in the infected host cells. We hypothesize that SPLUNC1 and NB co-localization is due to the GFP-tagged SPLUNC1 protein binding to the lipopolysaccharide (LPS) of the Gram-negative NB, which can play an important role in the host defense of nasopharyngeal epithelium. This research sheds new light on the mechanism of SPLUNC1 involvement in the host upper respiratory tract defense

  5. The effect of the cytoplasmic tail of influenza C virus CM2 protein on its biochemical properties and intracellular processing.

    Science.gov (United States)

    Shimotai, Yoshitaka; Goto, Takanari; Matsuzaki, Yoko; Muraki, Yasushi; Sugawara, Kanetsu; Hongo, Seiji

    2015-09-01

    CM2 is an integral membrane protein encoded by the influenza C virus M gene. To examine the effects of the cytoplasmic tail of CM2 on its biochemical properties, deletion and substitution mutations were introduced into CM2 cytoplasmic tail at residues 47-115, and the expressed CM2 mutants were investigated. Although the cytoplasmic tail is not essential for the oligomerization of CM2, it may affect the degree of oligomerization. The residues 47-48, 67-69, 73-90 and 113-115 were all required for the proper expression of CM2. Pulse-chase experiments suggest that residues 47-48, 67-69, 73-75 and 79-87 stabilize CM2, thereby affecting CM2 expression. The C-terminal region at residues 61-115 is not essential for CM2 transport to the cell surface, and a 14-amino-acid, but not an 11-amino-acid, cytoplasmic tail is sufficient for the cell surface expression of CM2. These results suggest that either certain amino acid sequences or the length of the CM2 cytoplasmic tail are necessary for the proper conformational maturation, stability, expression level and intracellular transport of CM2.

  6. The transmembrane channel-like protein family and human papillomaviruses

    Science.gov (United States)

    Horton, Jaime S; Stokes, Alexander J

    2014-01-01

    Epidermodysplasia verruciformis (EV) is a rare genodermatosis characterized by increased sensitivity to infection by the β-subtype of human papillomaviruses (β-HPVs), causing persistent, tinea versicolor-like dermal lesions. In a majority of affected individuals, these macular lesions progress to invasive cutaneous squamous cell carcinoma (CSCC) in sun-exposed areas. While mutations in transmembrane channel-like 6 (TMC6 / EVER1) and 8 (TMC8 / EVER2) have been causally linked to EV, their molecular functions are unclear. It is likely that their protective effects involve regulation of the β-HPV life cycle, host keratinocyte apoptosis vs. survival balance and/or T-cell interaction with infected host cells. PMID:24800179

  7. Imaging and structural studies of DNA–protein complexes and membrane ion channels

    KAUST Repository

    Marini, Monica

    2017-01-17

    In bio-imaging by electron microscopy, damage of the sample and limited contrast are the two main hurdles for reaching high image quality. We extend a new preparation method based on nanofabrication and super-hydrophobicity to the imaging and structural studies of nucleic acids, nucleic acid-protein complexes (DNA/Rad51 repair protein complex) and neuronal ion channels (gap-junction, K+ and GABA(A) channels) as paradigms of biological significance and increasing complexity. The preparation method is based on the liquid phase and is compatible with physiological conditions. Only in the very last stage, samples are dried for TEM analysis. Conventional TEM and high-resolution TEM (HRTEM) were used to achieve a resolution of 3.3 and 1.5 angstrom, respectively. The EM dataset quality allows the determination of relevant structural and metrological information on the DNA structure, DNA-protein interactions and ion channels, allowing the identification of specific macromolecules and their structure.

  8. Phg1/TM9 proteins control intracellular killing of bacteria by determining cellular levels of the Kil1 sulfotransferase in Dictyostelium.

    Directory of Open Access Journals (Sweden)

    Marion Le Coadic

    Full Text Available Dictyostelium discoideum has largely been used to study phagocytosis and intracellular killing of bacteria. Previous studies have shown that Phg1A, Kil1 and Kil2 proteins are necessary for efficient intracellular killing of Klebsiella bacteria. Here we show that in phg1a KO cells, cellular levels of lysosomal glycosidases and lysozyme are decreased, and lysosomal pH is increased. Surprisingly, overexpression of Kil1 restores efficient killing in phg1a KO cells without correcting these lysosomal anomalies. Conversely, kil1 KO cells are defective for killing, but their enzymatic content and lysosomal pH are indistinguishable from WT cells. The killing defect of phg1a KO cells can be accounted for by the observation that in these cells the stability and the cellular amount of Kil1 are markedly reduced. Since Kil1 is the only sulfotransferase characterized in Dictyostelium, an (unidentified sulfated factor, defective in both phg1a and kil1 KO cells, may play a key role in intracellular killing of Klebsiella bacteria. In addition, Phg1B plays a redundant role with Phg1A in controlling cellular amounts of Kil1 and intracellular killing. Finally, cellular levels of Kil1 are unaffected in kil2 KO cells, and Kil1 overexpression does not correct the killing defect of kil2 KO cells, suggesting that Kil2 plays a distinct role in intracellular killing.

  9. Functional fusions of T4 lysozyme in the third intracellular loop of a G protein-coupled receptor identified by a random screening approach in yeast.

    Science.gov (United States)

    Mathew, Elizabeth; Ding, Fa-Xiang; Naider, Fred; Dumont, Mark E

    2013-01-01

    The insertion of a stable soluble protein into loops of transmembrane proteins has proved to be a successful approach for enhancing their stabilities and crystallization, and may also be useful in contexts where the inserted proteins can modulate or report on the activities of membrane proteins. While the use of T4 lysozyme to replace portions of the third intracellular loops of G protein-coupled receptors (GPCRs) has allowed determination of the structures of members of this important class of receptors, the creation of such fusion proteins generally leads to loss of signaling function of the resulting fusion protein, since the third intracellular loops of GPCRs play critical roles in their interactions with G proteins. We describe here a random screening approach allowing insertion of T4 lysozyme into diverse positions in the third loop of the yeast α-pheromone receptor, a GPCR encoded by the yeast STE2 gene. Insertions were accompanied by varying extents of deletion or duplication of the loop. A set of phenotypic screens allow detection of potentially rare variant receptors that are expressed, bind to agonist and are capable of signal transduction via activation of the cognate G protein. A large fraction of screened full-length receptor variants containing at least partial duplications of the loop on either side of the inserted T4 lysozyme retain the ability to activate the downstream signaling pathway in response to binding of ligand. However, we were unable to identify any receptors with truncated C-termini that retain significant signaling function in the presence of inserted T4 lysozyme. Our results establish the feasibility of creating functional receptors containing insertions of T4 lysozyme in their third intracellular loops.

  10. Structure and inhibition of the SARS coronavirus envelope protein ion channel.

    Directory of Open Access Journals (Sweden)

    Konstantin Pervushin

    2009-07-01

    Full Text Available The envelope (E protein from coronaviruses is a small polypeptide that contains at least one alpha-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA, but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV that the transmembrane domain of E protein (ETM forms pentameric alpha-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular alpha-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293 cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA, but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.

  11. Yeast acyl-CoA-binding protein: acyl-CoA-binding affinity and effect on intracellular acyl-CoA pool size

    DEFF Research Database (Denmark)

    Knudsen, J; Faergeman, N J; Skøtt, H

    1994-01-01

    Acyl-CoA-binding protein (ACBP) is a 10 kDa protein characterized in vertebrates. We have isolated two ACBP homologues from the yeast Saccharomyces carlsbergensis, named yeast ACBP types 1 and 2. Both proteins contain 86 amino acid residues and are identical except for four conservative substitut...... resulted in a significant expansion of the intracellular acyl-CoA pool. Finally, Southern-blotting analysis of the two genes encoding ACBP types 1 and 2 in S. carlsbergensis strongly indicated that this species is a hybrid between S. cerevisiae and Saccharomyces monacensis....

  12. Modulation of Intracellular Quantum Dot to Fluorescent Protein Förster Resonance Energy Transfer via Customized Ligands and Spatial Control of Donor–Acceptor Assembly

    Directory of Open Access Journals (Sweden)

    Lauren D. Field

    2015-12-01

    Full Text Available Understanding how to controllably modulate the efficiency of energy transfer in Förster resonance energy transfer (FRET-based assemblies is critical to their implementation as sensing modalities. This is particularly true for sensing assemblies that are to be used as the basis for real time intracellular sensing of intracellular processes and events. We use a quantum dot (QD donor -mCherry acceptor platform that is engineered to self-assemble in situ wherein the protein acceptor is expressed via transient transfection and the QD donor is microinjected into the cell. QD-protein assembly is driven by metal-affinity interactions where a terminal polyhistidine tag on the protein binds to the QD surface. Using this system, we show the ability to modulate the efficiency of the donor–acceptor energy transfer process by controllably altering either the ligand coating on the QD surface or the precise location where the QD-protein assembly process occurs. Intracellularly, a short, zwitterionic ligand mediates more efficient FRET relative to longer ligand species that are based on the solubilizing polymer, poly(ethylene glycol. We further show that a greater FRET efficiency is achieved when the QD-protein assembly occurs free in the cytosol compared to when the mCherry acceptor is expressed tethered to the inner leaflet of the plasma membrane. In the latter case, the lower FRET efficiency is likely attributable to a lower expression level of the mCherry acceptor at the membrane combined with steric hindrance. Our work points to some of the design considerations that one must be mindful of when developing FRET-based sensing schemes for use in intracellular sensing.

  13. Membrane Incorporation, Channel Formation, and Disruption of Calcium Homeostasis by Alzheimer's β-Amyloid Protein

    Directory of Open Access Journals (Sweden)

    Masahiro Kawahara

    2011-01-01

    Full Text Available Oligomerization, conformational changes, and the consequent neurodegeneration of Alzheimer's β-amyloid protein (AβP play crucial roles in the pathogenesis of Alzheimer's disease (AD. Mounting evidence suggests that oligomeric AβPs cause the disruption of calcium homeostasis, eventually leading to neuronal death. We have demonstrated that oligomeric AβPs directly incorporate into neuronal membranes, form cation-sensitive ion channels (“amyloid channels”, and cause the disruption of calcium homeostasis via the amyloid channels. Other disease-related amyloidogenic proteins, such as prion protein in prion diseases or α-synuclein in dementia with Lewy bodies, exhibit similarities in the incorporation into membranes and the formation of calcium-permeable channels. Here, based on our experimental results and those of numerous other studies, we review the current understanding of the direct binding of AβP into membrane surfaces and the formation of calcium-permeable channels. The implication of composition of membrane lipids and the possible development of new drugs by influencing membrane properties and attenuating amyloid channels for the treatment and prevention of AD is also discussed.

  14. Sendai virus C protein plays a role in restricting PKR activation by limiting the generation of intracellular double-stranded RNA.

    Science.gov (United States)

    Takeuchi, Kenji; Komatsu, Takayuki; Kitagawa, Yoshinori; Sada, Kiyonao; Gotoh, Bin

    2008-10-01

    Sendai virus (SeV) C protein is a multifunctional protein that plays important roles in regulating viral genome replication and transcription, antagonizing the host interferon system, suppressing virus-induced apoptosis, and facilitating virus assembly and budding. We here report a novel role of SeV C protein, the limitation of double-stranded RNA (dsRNA) generation for maintaining the rate of protein synthesis in infected cells. It was found that the intracellular protein synthesis rate was maintained even after wild-type (wt) SeV infection, but markedly suppressed following C-knockout SeV infection. This indicates the requirement of C protein for maintaining protein synthesis after infection. In contrast to wt SeV infection, C-knockout SeV infection caused phosphorylation of both the translation initiation factor eIF2alpha and dsRNA-dependent protein kinase (PKR). Phosphorylation of eIF2alpha occurred mainly due to the action of PKR, since knockdown of PKR by small interfering RNA limited eIF2alpha phosphorylation. C protein, however, could inhibit neither poly(I):poly(C)-activated nor Newcastle disease virus-induced phosphorylation of PKR and eIF2alpha, suggesting that C protein does not target common pathways leading to PKR activation. Immunofluorescent staining experiments with a monoclonal antibody specifically recognizing dsRNA revealed generation of a large amount of dsRNA in cells infected with C-knockout SeV but not wt SeV. The dsRNA generation as well as phosphorylation of PKR and eIF2alpha induced by C-knockout SeV was markedly suppressed in cells constitutively expressing C protein. Taken together, these results demonstrate that the SeV C protein limits generation of dsRNA, thereby keeping PKR inactive to maintain intracellular protein synthesis.

  15. The Escherichia coli small protein MntS and exporter MntP optimize the intracellular concentration of manganese.

    Directory of Open Access Journals (Sweden)

    Julia E Martin

    2015-03-01

    Full Text Available Escherichia coli does not routinely import manganese, but it will do so when iron is unavailable, so that manganese can substitute for iron as an enzyme cofactor. When intracellular manganese levels are low, the cell induces the MntH manganese importer plus MntS, a small protein of unknown function; when manganese levels are high, the cell induces the MntP manganese exporter and reduces expression of MntH and MntS. The role of MntS has not been clear. Previous work showed that forced MntS synthesis under manganese-rich conditions caused bacteriostasis. Here we find that when manganese is scarce, MntS helps manganese to activate a variety of enzymes. Its overproduction under manganese-rich conditions caused manganese to accumulate to very high levels inside the cell; simultaneously, iron levels dropped precipitously, apparently because manganese-bound Fur blocked the production of iron importers. Under these conditions, heme synthesis stopped, ultimately depleting cytochrome oxidase activity and causing the failure of aerobic metabolism. Protoporphyrin IX accumulated, indicating that the combination of excess manganese and iron deficiency had stalled ferrochelatase. The same chain of events occurred when mutants lacking MntP, the manganese exporter, were exposed to manganese. Genetic analysis suggested the possibility that MntS exerts this effect by inhibiting MntP. We discuss a model wherein during transitions between low- and high-manganese environments E. coli uses MntP to compensate for MntH overactivity, and MntS to compensate for MntP overactivity.

  16. Paraquat Resistant1, a Golgi-localized putative transporter protein, is involved in intracellular transport of paraquat.

    Science.gov (United States)

    Li, Jianyong; Mu, Jinye; Bai, Jiaoteng; Fu, Fuyou; Zou, Tingting; An, Fengying; Zhang, Jian; Jing, Hongwei; Wang, Qing; Li, Zhen; Yang, Shuhua; Zuo, Jianru

    2013-05-01

    Paraquat is one of the most widely used herbicides worldwide. In green plants, paraquat targets the chloroplast by transferring electrons from photosystem I to molecular oxygen to generate toxic reactive oxygen species, which efficiently induce membrane damage and cell death. A number of paraquat-resistant biotypes of weeds and Arabidopsis (Arabidopsis thaliana) mutants have been identified. The herbicide resistance in Arabidopsis is partly attributed to a reduced uptake of paraquat through plasma membrane-localized transporters. However, the biochemical mechanism of paraquat resistance remains poorly understood. Here, we report the identification and characterization of an Arabidopsis paraquat resistant1 (par1) mutant that shows strong resistance to the herbicide without detectable developmental abnormalities. PAR1 encodes a putative l-type amino acid transporter protein localized to the Golgi apparatus. Compared with the wild-type plants, the par1 mutant plants show similar efficiency of paraquat uptake, suggesting that PAR1 is not directly responsible for the intercellular uptake of paraquat. However, the par1 mutation caused a reduction in the accumulation of paraquat in the chloroplast, suggesting that PAR1 is involved in the intracellular transport of paraquat into the chloroplast. We identified a PAR1-like gene, OsPAR1, in rice (Oryza sativa). Whereas the overexpression of OsPAR1 resulted in hypersensitivity to paraquat, the knockdown of its expression using RNA interference conferred paraquat resistance on the transgenic rice plants. These findings reveal a unique mechanism by which paraquat is actively transported into the chloroplast and also provide a practical approach for genetic manipulations of paraquat resistance in crops.

  17. Energy transduction and signal averaging of fluctuating electric fields by a single protein ion channel.

    Science.gov (United States)

    Verdia-Baguena, C; Gomez, V; Cervera, J; Ramirez, P; Mafe, S

    2016-12-21

    We demonstrate the electrical rectification and signal averaging of fluctuating signals using a biological nanostructure in aqueous solution: a single protein ion channel inserted in the lipid bilayer characteristic of cell membranes. The conversion of oscillating, zero time-average potentials into directional currents permits charging of a load capacitor to significant steady-state voltages within a few minutes in the case of the outer membrane porin F (OmpF) protein, a bacterial channel of Escherichia coli. The experiments and simulations show signal averaging effects at a more fundamental level than the traditional cell and tissue scales, which are characterized by ensembles of many ion channels operating simultaneously. The results also suggest signal transduction schemes with bio-electronic interfaces and ionic circuits where soft matter nanodiodes can be coupled to conventional electronic elements.

  18. Engineering of an E. coli outer membrane protein FhuA with increased channel diameter

    Directory of Open Access Journals (Sweden)

    Dworeck Tamara

    2011-08-01

    Full Text Available Abstract Background Channel proteins like FhuA can be an alternative to artificial chemically synthesized nanopores. To reach such goals, channel proteins must be flexible enough to be modified in their geometry, i.e. length and diameter. As continuation of a previous study in which we addressed the lengthening of the channel, here we report the increasing of the channel diameter by genetic engineering. Results The FhuA Δ1-159 diameter increase has been obtained by doubling the amino acid sequence of the first two N-terminal β-strands, resulting in variant FhuA Δ1-159 Exp. The total number of β-strands increased from 22 to 24 and the channel surface area is expected to increase by ~16%. The secondary structure analysis by circular dichroism (CD spectroscopy shows a high β-sheet content, suggesting the correct folding of FhuA Δ1-159 Exp. To further prove the FhuA Δ1-159 Exp channel functionality, kinetic measurement using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine were conducted. The results indicated a 17% faster diffusion kinetic for FhuA Δ1-159 Exp as compared to FhuA Δ1-159, well correlated to the expected channel surface area increase of ~16%. Conclusion In this study using a simple "semi rational" approach the FhuA Δ1-159 diameter was enlarged. By combining the actual results with the previous ones on the FhuA Δ1-159 lengthening a new set of synthetic nanochannels with desired lengths and diameters can be produced, broadening the FhuA Δ1-159 applications. As large scale protein production is possible our approach can give a contribution to nanochannel industrial applications.

  19. Analysis of the intracellular localization of p73 N-terminal protein isoforms TAp73 and ∆Np73 in medulloblastoma cell lines.

    Science.gov (United States)

    Nekulová, Marta; Zitterbart, Karel; Sterba, Jaroslav; Veselská, Renata

    2010-10-01

    The protein homologous to the tumor suppressor p53, p73, has essential roles in development and tumorigenesis. This protein exists in a wide range of isoforms with different, even antagonistic, functions. However, there are virtually no detailed morphological studies analyzing the endogenous expression of p73 isoforms at the cellular level in cancer cells. In this study, we investigated the expression and subcellular distribution of two N-terminal isoforms, TAp73 and ΔNp73, in medulloblastoma cells using immunofluorescence microscopy. Both proteins were observed in all cell lines examined, but differences were noted in their intracellular localization between the reference Daoy cell line and four newly established medulloblastoma cell lines (MBL-03, MBL-06, MBL-07 and MBL-10). In the new cell lines, TAp73 and ΔNp73 were located predominantly in cell nuclei. However, there was heterogeneity in TAp73 distribution in the cells of all MBL cell lines, with the protein located in the nucleus and also in a limited non-random area in the cytoplasm. In a small percentage of cells, we detected cytoplasmic localization of TAp73 only, i.e., nuclear exclusion was observed. Our results provide a basis for future studies on the causes and function of distinct intracellular localization of p73 protein isoforms with respect to different protein-protein interactions in medulloblastoma cells.

  20. G Protein Regulation of Neuronal Calcium Channels: Back to the Future

    Czech Academy of Sciences Publication Activity Database

    Proft, Juliane; Weiss, Norbert

    2015-01-01

    Roč. 87, č. 6 (2015), s. 890-906 ISSN 0026-895X R&D Projects: GA ČR GA15-13556S Institutional support: RVO:61388963 Keywords : voltage gated calcium channels Cav * G proteins * GPCR Subject RIV: CE - Biochemistry Impact factor: 3.931, year: 2015

  1. KCNQ1 channel modulation by KCNE proteins via the voltage-sensing domain.

    Science.gov (United States)

    Nakajo, Koichi; Kubo, Yoshihiro

    2015-06-15

    The gating of the KCNQ1 potassium channel is drastically regulated by auxiliary subunit KCNE proteins. KCNE1, for example, slows the activation kinetics of KCNQ1 by two orders of magnitude. Like other voltage-gated ion channels, the opening of KCNQ1 is regulated by the voltage-sensing domain (VSD; S1-S4 segments). Although it has been known that KCNE proteins interact with KCNQ1 via the pore domain, some recent reports suggest that the VSD movement may be altered by KCNE. The altered VSD movement of KCNQ1 by KCNE proteins has been examined by site-directed mutagenesis, the scanning cysteine accessibility method (SCAM), voltage clamp fluorometry (VCF) and gating charge measurements. These accumulated data support the idea that KCNE proteins interact with the VSDs of KCNQ1 and modulate the gating of the KCNQ1 channel. In this review, we will summarize recent findings and current views of the KCNQ1 modulation by KCNE via the VSD. In this context, we discuss our recent findings that KCNE1 may alter physical interactions between the S4 segment (VSD) and the S5 segment (pore domain) of KCNQ1. Based on these findings from ourselves and others, we propose a hypothetical mechanism for how KCNE1 binding alters the VSD movement and the gating of the channel. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  2. Intracellular formation of α-synuclein oligomers and the effect of heat shock protein 70 characterized by confocal single particle spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Levin, Johannes [Department of Neurology, Ludwig-Maximilians-University, Marchioninistr. 15, 81377 Munich (Germany); German Center for Neurodegenerative Diseases – DZNE, Site Munich, Feodor-Lynen-Str. 17, 81377 Munich (Germany); Hillmer, Andreas S. [Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Feodor-Lynen-Str. 23, 81377 Munich (Germany); Högen, Tobias [Department of Neurology, Ludwig-Maximilians-University, Marchioninistr. 15, 81377 Munich (Germany); McLean, Pamela J. [Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224 (United States); Giese, Armin, E-mail: armin.giese@med.uni-muenchen.de [Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Feodor-Lynen-Str. 23, 81377 Munich (Germany)

    2016-08-12

    Synucleinopathies such as dementia with Lewy bodies or Parkinson’s disease are characterized by intracellular deposition of pathologically aggregated α-synuclein. The details of the molecular pathogenesis of PD and especially the conditions that lead to intracellular aggregation of α-synuclein and the role of these aggregates in cell death remain unknown. In cell free in vitro systems considerable knowledge about the aggregation processes has been gathered. In comparison, the knowledge about these aggregation processes in cells is far behind. In cells α-synuclein aggregates can be toxic. However, the crucial particle species responsible for decisive steps in pathogenesis such as seeding a continuing aggregation process and triggering cell death remain to be identified. In order to understand the complex nature of intracellular α-synuclein aggregate formation, we analyzed fluorescent particles formed by venus and α-synuclein-venus fusion proteins and α-synuclein-hemi-venus fusion proteins derived from gently lyzed cells. With these techniques we were able to identify and characterize α-synuclein oligomers formed in cells. Especially the use of α-synuclein-hemi-venus fusion proteins enabled us to identify very small α-synuclein oligomers with high sensitivity. Furthermore, we were able to study the molecular effect of heat shock protein 70, which is known to inhibit α-synuclein aggregation in cells. Heat shock protein 70 does not only influence the size of α-synuclein oligomers, but also their quantity. In summary, this approach based on fluorescence single particle spectroscopy, that is suited for high throughput measurements, can be used to detect and characterize intracellularly formed α-synuclein aggregates and characterize the effect of molecules that interfere with α-synuclein aggregate formation. - Highlights: • Single particle spectroscopy detects intracellular formed α-synuclein aggregates. • Fusion proteins allow detection of protein

  3. Modifications in host cell cytoskeleton structure and function mediated by intracellular HIV-1 Tat protein are greatly dependent on the second coding exon

    OpenAIRE

    López-Huertas, María Rosa; Callejas, Sergio; Abia, David; Mateos, Elena; Dopazo, Ana; Alcamí, Antonio; Coiras, Mayte

    2010-01-01

    The human immunodeficiency virus type 1 (HIV-1) regulator Tat is essential for viral replication because it achieves complete elongation of viral transcripts. Tat can be released to the extracellular space and taken up by adjacent cells, exerting profound cytoskeleton rearrangements that lead to apoptosis. In contrast, intracellular Tat has been described as protector from apoptosis. Tat gene is composed by two coding exons that yield a protein of 101 amino acids (a...

  4. Disruption of Intracellular ATP Generation and Tight Junction Protein Expression during the Course of Brain Edema Induced by Subacute Poisoning of 1,2-Dichloroethane

    Directory of Open Access Journals (Sweden)

    Gaoyang Wang

    2018-01-01

    Full Text Available The aim of this study was to explore changes in intracellular ATP generation and tight junction protein expression during the course of brain edema induced by subacute poisoning of 1,2-dichloroethane (1,2-DCE. Mice were exposed to 1.2 g/m3 1,2-DCE for 3.5 h per day for 1, 2, or 3 days, namely group A, B, and C. Na+-K+-ATPase and Ca2+-ATPase activity, ATP and lactic acid content, intracellular free Ca2+ concentration and ZO-1 and occludin expression in the brain were measured. Results of present study disclosed that Ca2+-ATPase activities in group B and C, and Na+/K+-ATPase activity in group C decreased, whereas intracellular free Ca2+ concentrations in group B and C increased significantly compared with control. Moreover, ATP content decreased, whereas lactic acid content increased significantly in group C compared with control. On the other hand, expressions of ZO-1 and occludin at both the protein and gene levels in group B and C decreased significantly compared with control. In conclusion, findings from this study suggest that calcium overload and depressed expression of tight junction associated proteins, such as ZO-1 and occludin might play an important role in the early phase of brain edema formation induced by subacute poisoning of 1,2-DCE.

  5. Molecular Characterization of LRB7 Gene and a Water Channel Protein TIP2 in Chorispora bungeana

    Science.gov (United States)

    Liang, Zhaoxu; Di, Cuixia; Fang, Weikuan; Wu, Kaichao; Chen, Maoshan; He, Shanshan; Zeng, Yuan; Jing, Yan; Liang, Jun; Tan, Fang; Li, Song; Chen, Tuo; Liu, Guangxiu

    2016-01-01

    Background. Water channel proteins, also called aquaporins, are integral membrane proteins from major intrinsic protein (MIP) family and involved in several pathways including not only water transport but also cell signaling, reproduction, and photosynthesis. The full cDNA and protein sequences of aquaporin in Chorispora bungeana Fisch. & C.A. Mey (C. bungeana) are still unknown. Results. In this study, PCR and rapid amplification of cDNA ends approaches were used to clone the full cDNA of LRB7 (GenBank accession number: EU636988) of C. bungeana. Sequence analysis indicated that it was 1235 bp, which had two introns and encoded a protein of 250 amino acids. Structure analysis revealed that the protein had two conserved NPA motifs, one of which is MIP signature sequence (SGxHxNPAVT), six membrane helix regions, and additional membrane-embedded domains. Phylogenetic analysis suggested that the protein was from TIP2 subgroup. Surprisingly, semiquantitative RT-PCR experiment and western blot analysis showed that LRB7 and TIP2 were only detectable in roots, unlike Arabidopsis and Raphanus. Connecting with our previous studies, LRB7 was supported to associate with chilling-tolerance in C. bungeana. Conclusion. This is the first time to characterize the full sequences of LRB7 gene and water channel protein in C. bungeana. Our findings contribute to understanding the water transports in plants under low temperatures. PMID:27689074

  6. Molecular Characterization of LRB7 Gene and a Water Channel Protein TIP2 in Chorispora bungeana

    Directory of Open Access Journals (Sweden)

    Ming Li

    2016-01-01

    Full Text Available Background. Water channel proteins, also called aquaporins, are integral membrane proteins from major intrinsic protein (MIP family and involved in several pathways including not only water transport but also cell signaling, reproduction, and photosynthesis. The full cDNA and protein sequences of aquaporin in Chorispora bungeana Fisch. & C.A. Mey (C. bungeana are still unknown. Results. In this study, PCR and rapid amplification of cDNA ends approaches were used to clone the full cDNA of LRB7 (GenBank accession number: EU636988 of C. bungeana. Sequence analysis indicated that it was 1235 bp, which had two introns and encoded a protein of 250 amino acids. Structure analysis revealed that the protein had two conserved NPA motifs, one of which is MIP signature sequence (SGxHxNPAVT, six membrane helix regions, and additional membrane-embedded domains. Phylogenetic analysis suggested that the protein was from TIP2 subgroup. Surprisingly, semiquantitative RT-PCR experiment and western blot analysis showed that LRB7 and TIP2 were only detectable in roots, unlike Arabidopsis and Raphanus. Connecting with our previous studies, LRB7 was supported to associate with chilling-tolerance in C. bungeana. Conclusion. This is the first time to characterize the full sequences of LRB7 gene and water channel protein in C. bungeana. Our findings contribute to understanding the water transports in plants under low temperatures.

  7. High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter

    Directory of Open Access Journals (Sweden)

    D’Urzo Nunzia

    2013-02-01

    Full Text Available Abstract Background In past years research has focused on the development of alternative Gram positive bacterial expression systems to produce industrially relevant proteins. Brevibacillus choshinensis is an easy to handle non-sporulating bacterium, lacking extracellular proteases, that has been already shown to provide a high level of recombinant protein expression. One major drawback, limiting the applicability of the Brevibacillus expression system, is the absence of expression vectors based on inducible promoters. Here we used the PxylA inducible promoter, commonly employed in other Bacillae expression systems, in Brevibacillus. Results Using GFP, α-amylase and TcdA-GT as model proteins, high level of intracellular protein expression (up to 250 mg/L for the GFP was achieved in Brevibacillus, using the pHis1522 vector carrying the B. megaterium xylose-inducible promoter (PxylA. The GFP expression yields were more than 25 fold higher than those reported for B. megaterium carrying the same vector. All the tested proteins show significant increment in their expression levels (2-10 folds than those obtained using the available plasmids based on the P2 constitutive promoter. Conclusion Combining the components of two different commercially available Gram positive expression systems, such as Brevibacillus (from Takara Bio and B. megaterium (from Mobitec, we demonstrate that vectors based on the B. megaterium PxylA xylose inducible promoter can be successfully used to induce high level of intracellular expression of heterologous proteins in Brevibacillus.

  8. The small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties

    International Nuclear Information System (INIS)

    Lee, Changhee; Yoo, Dongwan

    2006-01-01

    The small envelope (E) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is a hydrophobic 73 amino acid protein encoded in the internal open reading frame (ORF) of the bicistronic mRNA2. As a first step towards understanding the biological role of E protein during PRRSV replication, E gene expression was blocked in a full-length infectious clone by mutating the ATG translational initiation to GTG, such that the full-length mutant genomic clone was unable to synthesize the E protein. DNA transfection of PRRSV-susceptible cells with the E gene knocked-out genomic clone showed the absence of virus infectivity. P129-ΔE-transfected cells however produced virion particles in the culture supernatant, and these particles contained viral genomic RNA, demonstrating that the E protein is essential for PRRSV infection but dispensable for virion assembly. Electron microscopy suggests that the P129-ΔE virions assembled in the absence of E had a similar appearance to the wild-type particles. Strand-specific RT-PCR demonstrated that the E protein-negative, non-infectious P129-ΔE virus particles were able to enter cells but further steps of replication were interrupted. The entry of PRRSV has been suggested to be via receptor-mediated endocytosis, and lysomotropic basic compounds and known ion-channel blocking agents both inhibited PRRSV replication effectively during the uncoating process. The expression of E protein in Escherichia coli-mediated cell growth arrests and increased the membrane permeability. Cross-linking experiments in cells infected with PRRSV or transfected with E gene showed that the E protein was able to form homo-oligomers. Taken together, our data suggest that the PRRSV E protein is likely an ion-channel protein embedded in the viral envelope and facilitates uncoating of virus and release of the genome in the cytoplasm

  9. From GTP and G proteins to TRPC channels: a personal account.

    Science.gov (United States)

    Birnbaumer, Lutz

    2015-09-01

    By serendipity and good fortune, as a postdoctoral fellow in 1967, I landed at the right place at the right time, as I was allowed to investigate the mechanism by which hormones activate the enzyme adenylyl cyclase (then adenyl cyclase) in Martin Rodbell's Laboratory at the NIH in Bethesda, Maryland. The work uncovered first, the existence of receptors separate from the enzyme and then, the existence of transduction mechanisms requiring guanosine-5'-triphosphate (GTP) and Mg(2+). With my laboratory colleagues first and postdoctoral fellows after leaving NIH, I participated in the development of the field "signal transduction by G proteins," uncovered by molecular cloning several G-protein-coupled receptors (GPCRs) and became interested in both the molecular makeup of voltage-gated Ca channels and Ca2+ homeostasis downstream of activation of phospholipase C (PLC) by the Gq/11 signaling pathway. We were able to confirm the hypothesis that there would be mammalian homologues of the Drosophila "transient receptor potential" channel and discovered the existence of six of the seven mammalian genes, now called transient receptor potential canonical (TRPC) channels. In the present article, I summarize from a bird's eye view of what I feel were key findings along this path, not only from my laboratory but also from many others, that allowed for the present knowledge of cell signaling involving G proteins to evolve. Towards the end, I summarize roles of TRPC channels in health and disease.

  10. Selenium potentiates the anticancer effect of cisplatin against oxidative stress and calcium ion signaling-induced intracellular toxicity in MCF-7 breast cancer cells: involvement of the TRPV1 channel.

    Science.gov (United States)

    Sakallı Çetin, Esin; Nazıroğlu, Mustafa; Çiğ, Bilal; Övey, İshak Suat; Aslan Koşar, Pınar

    2017-02-01

    In breast cancers, calcium signaling is a main cause of proliferation and apoptosis of breast cancer cells. Although previous studies have implicated the transient receptor potential vanilloid 1 (TRPV1) cation channel, the synergistic inhibition effects of selenium (Se) and cisplatin in cancer and the suppression of ongoing apoptosis have not yet been investigated in MCF-7 breast cancer cells. This study investigates the anticancer properties of Se through TRPV1 channel activity in MCF-7 breast cancer cell line cultures when given alone or in combination with cisplatin. The MCF-7 cells were divided into four groups: the control group, the Se-treated group (200 nM), the cisplatin-treated group (40 μM) and the Se + cisplatin-treated group. The intracellular free calcium ion concentration and current densities increased with TRPV1 channel activator capsaicin (0.01 mM), but they decreased with the TRPV1 blocker capsazepine (0.1 mM), Se, cisplatin, and Se + cisplatin incubations. However, mitochondrial membrane depolarization, apoptosis, and the caspase 3, and caspase 9 values increased in the Se-treated group and the cisplatin-treated group, although Western blot (procaspase 3 and 9) results and the cell viability levels decreased with the Se and Se + cisplatin treatments. Apoptosis and caspase-3 were further increased with the Se + cisplatin treatment. Intracellular reactive oxygen species production increased with the cisplatin treatment, but not with the Se treatment. This study's results report, for the first time, that at a cellular level, Se and cisplatin interact on the same intracellular toxic cascade, and the combination of these two drugs can result in a remarkable anticancer effect through modulation of the TRPV1.

  11. The cannabinoid agonist WIN55,212-2 increases intracellular calcium via CB1 receptor coupling to Gq/11 G proteins.

    Science.gov (United States)

    Lauckner, Jane E; Hille, Bertil; Mackie, Ken

    2005-12-27

    Central nervous system responses to cannabis are primarily mediated by CB(1) receptors, which couple preferentially to G(i/o) G proteins. Here, we used calcium photometry to monitor the effect of CB(1) activation on intracellular calcium concentration. Perfusion with 5 microM CB(1) aminoalkylindole agonist, WIN55,212-2 (WIN), increased intracellular calcium by several hundred nanomolar in human embryonic kidney 293 cells stably expressing CB(1) and in cultured hippocampal neurons. The increase was blocked by coincubation with the CB(1) antagonist, SR141716A, and was absent in nontransfected human embryonic kidney 293 cells. The calcium rise was WIN-specific, being essentially absent in cells treated with other classes of cannabinoid agonists, including Delta(9)-tetrahydrocannabinol, HU-210, CP55,940, 2-arachidonoylglycerol, methanandamide, and cannabidiol. The increase in calcium elicited by WIN was independent of G(i/o), because it was present in pertussis toxin-treated cells. Indeed, pertussis toxin pretreatment enhanced the potency and efficacy of WIN to increase intracellular calcium. The calcium increases appeared to be mediated by G(q) G proteins and phospholipase C, because they were markedly attenuated in cells expressing dominant-negative G(q) or treated with the phospholipase C inhibitors U73122 and ET-18-OCH(3) and were accompanied by an increase in inositol phosphates. The calcium increase was blocked by the sarco/endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin, the inositol trisphosphate receptor inhibitor xestospongin D, and the ryanodine receptor inhibitors dantrolene and 1,1'-diheptyl-4,4'-bipyridinium dibromide, but not by removal of extracellular calcium, showing that WIN releases calcium from intracellular stores. In summary, these results suggest that WIN stabilizes CB(1) receptors in a conformation that enables G(q) signaling, thus shifting the G protein specificity of the receptor.

  12. Isolation and identification of a sodium channel-inhibiting protein from eggs of black widow spiders.

    Science.gov (United States)

    Li, Jianjun; Yan, Yizhong; Yu, Hai; Peng, Xiaozhen; Zhang, Yiya; Hu, Weijun; Duan, Zhigui; Wang, Xianchun; Liang, Songping

    2014-04-01

    The eggs of black widow spider (L. tredecimguttatus) have been demonstrated to be rich in biologically active components that exhibit great research value and application foreground. In the present study, a protein toxin, named Latroeggtoxin-II, was isolated from the eggs using the combination of gel filtration, ion exchange chromatography and reversed-phase high performance liquid chromatography. Electrospray mass spectrometric analysis indicated that the molecular weight of the protein was 28.69 kDa, and Edman degradation revealed that its N-terminal sequence was ESIQT STYVP NTPNQ KFDYE VGKDY-. After being abdominally injected into mice and P. americana, the protein could make the animals especially P. americana display a series of poisoning symptoms. Electrophysiological experiments demonstrated that the protein could selectively inhibit tetrodotoxin-resistant Na(+) channel currents in rat dorsal root ganglion neurons, without significant effect on the tetrodotoxin-sensitive Na(+) channel currents. Using multiple proteomic strategies, the purified protein was shown to have only a few similarities to the existing proteins in the databases, suggesting that it was a novel protein isolated from the eggs of black widow spiders. Copyright © 2014. Published by Elsevier B.V.

  13. Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing.

    Science.gov (United States)

    Elaswad, Ahmed; Khalil, Karim; Cline, David; Page-McCaw, Patrick; Chen, Wenbiao; Michel, Maximilian; Cone, Roger; Dunham, Rex

    2018-01-20

    The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

  14. Do cysteine residues regulate transient receptor potential canonical type 6 (TRPC6) channel protein expression?

    DEFF Research Database (Denmark)

    Thilo, Florian; Liu, Ying; Krueger, Katharina

    2012-01-01

    The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed that patie......The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed...... that patients with chronic renal failure had significantly elevated homocysteine levels and TRPC6 mRNA expression levels in monocytes compared to control subjects. We further observed that administration of homocysteine or acetylcysteine significantly increased TRPC6 channel protein expression compared...

  15. Increasing the production yield of recombinant protein in transgenic seeds by expanding the deposition space within the intracellular compartment

    OpenAIRE

    Takaiwa, Fumio

    2013-01-01

    Seeds must maintain a constant level of nitrogen in order to germinate. When recombinant proteins are produced while endogenous seed protein expression is suppressed, the production levels of the foreign proteins increase to compensate for the decreased synthesis of endogenous proteins. Thus, exchanging the production of endogenous seed proteins for that of foreign proteins is a promising approach to increase the yield of foreign recombinant proteins. Providing a space for the deposition of r...

  16. Establishing homology between mitochondrial calcium uniporters, prokaryotic magnesium channels and chlamydial IncA proteins.

    Science.gov (United States)

    Lee, Andre; Vastermark, Ake; Saier, Milton H

    2014-08-01

    Mitochondrial calcium uniporters (MCUs) (TC no. 1.A.77) are oligomeric channel proteins found in the mitochondrial inner membrane. MCUs have two well-conserved transmembrane segments (TMSs), connected by a linker, similar to bacterial MCU homologues. These proteins and chlamydial IncA proteins (of unknown function; TC no. 9.B.159) are homologous to prokaryotic Mg(2+) transporters, AtpI and AtpZ, based on comparison scores of up to 14.5 sds. A phylogenetic tree containing all of these proteins showed that the AtpZ proteins cluster coherently as a subset within the large and diverse AtpI cluster, which branches separately from the MCUs and IncAs, both of which cluster coherently. The MCUs and AtpZs share the same two TMS topology, but the AtpIs have four TMSs, and IncAs can have either two (most frequent) or four (less frequent) TMSs. Binary alignments, comparison scores and motif analyses showed that TMSs 1 and 2 align with TMSs 3 and 4 of the AtpIs, suggesting that the four TMS AtpI proteins arose via an intragenic duplication event. These findings establish an evolutionary link interconnecting eukaryotic and prokaryotic Ca(2+) and Mg(2+) transporters with chlamydial IncAs, and lead us to suggest that all members of the MCU superfamily, including IncAs, function as divalent cation channels. © 2014 The Authors.

  17. Intracellular glutathione production, but not protein glycation, underlies the protective effects of captopril against 2-deoxy-D-ribose-induced β-cell damage.

    Science.gov (United States)

    Koh, Gwanpyo; Yang, Eun-Jin; Kim, Ji Young; Hyun, Jonghoon; Yoo, Soyeon; Lee, Sang Ah

    2015-10-01

    Our previous study reported that both oxidative stress and protein glycation were the principal mechanisms underlying 2‑deoxy‑D‑ribose (dRib)‑induced pancreatic β‑cell damage. The aim of the present study was to investigate the effects of captopril on dRib‑induced damage in pancreatic β‑cells, as well as to determine the mechanisms underlying these effects. Treatment with dRib increased the levels of cytotoxicity, apoptosis, and intracellular reactive oxygen species in Syrian hamster insulinoma HIT‑T15 cells; however, pretreatment with captopril significantly inhibited the effects of dRib. The intracellular levels of reduced and oxidized glutathione were depleted following treatment with dRib; however, these levels were restored following HIT‑T15 cell treatment with captopril. In rat islets, dRib stimulation suppressed the mRNA expression levels of insulin, and pancreatic and duodenal homeobox 1, as well as insulin content; however, these effects were dose‑dependently reversed by treatment with captopril. Treatment with buthionine sulfoximine, an inhibitor of intracellular glutathione biosynthesis, inhibited the protective effects of captopril on dRib‑mediated glutathione depletion and cytotoxicity in HIT‑T15 cells. Following incubation with albumin, dRib increased the formation of dicarbonyl and advanced glycation end products. Treatment with captopril did not inhibit the dRib‑induced increase in production of dicarbonyl and advanced glycation end products. In conclusion, treatment with captopril reversed dRib‑induced oxidative damage and suppression of insulin expression in β‑cells. The mechanism underlying the protective effects of captopril may involve increased intracellular glutathione production, rather than protein glycation.

  18. An efficient method for modeling kinetic behavior of channel proteins in cardiomyocytes.

    Science.gov (United States)

    Wang, Chong; Beyerlein, Peter; Pospisil, Heike; Krause, Antje; Nugent, Chris; Dubitzky, Werner

    2012-01-01

    Characterization of the kinetic and conformational properties of channel proteins is a crucial element in the integrative study of congenital cardiac diseases. The proteins of the ion channels of cardiomyocytes represent an important family of biological components determining the physiology of the heart. Some computational studies aiming to understand the mechanisms of the ion channels of cardiomyocytes have concentrated on Markovian stochastic approaches. Mathematically, these approaches employ Chapman-Kolmogorov equations coupled with partial differential equations. As the scale and complexity of such subcellular and cellular models increases, the balance between efficiency and accuracy of algorithms becomes critical. We have developed a novel two-stage splitting algorithm to address efficiency and accuracy issues arising in such modeling and simulation scenarios. Numerical experiments were performed based on the incorporation of our newly developed conformational kinetic model for the rapid delayed rectifier potassium channel into the dynamic models of human ventricular myocytes. Our results show that the new algorithm significantly outperforms commonly adopted adaptive Runge-Kutta methods. Furthermore, our parallel simulations with coupled algorithms for multicellular cardiac tissue demonstrate a high linearity in the speedup of large-scale cardiac simulations.

  19. The effects of changes in glutathione levels through exogenous agents on intracellular cysteine content and protein adduct formation in chronic alcohol-treated VL17A cells.

    Science.gov (United States)

    Kumar, S Mathan; Haridoss, Madhumitha; Swaminathan, Kavitha; Gopal, Ramesh Kumar; Clemens, Dahn; Dey, Aparajita

    2017-02-01

    Alcohol-mediated liver injury is associated with changes in the level of the major cellular antioxidant glutathione (GSH). It is interesting to investigate if the changes in intracellular GSH level through exogenous agents affect the intracellular cysteine content and the protein adduct formation indicative of oxidative insult in chronic alcohol treated liver cells. In VL-17A cells treated with 2 mM N-acetyl cysteine (NAC) or 0.1 mM ursodeoxycholic acid (UDCA) plus 100 mM ethanol, an increase in cysteine concentration which was accompanied by decreases in hydroxynonenal (HNE) and glutathionylated protein adducts were observed. Pretreatment of 100 mM ethanol treated VL-17A cells with 0.4 mM buthionine sulfoximine (BSO) or 1 mM diethyl maleate (DEM) had opposite effects. Thus, altered GSH level through exogenous agents may either potentiate or ameliorate chronic alcohol-mediated protein adduct formation and change the cysteine level in chronic alcohol treated VL-17A cells. The gene expression of non-treated and ethanol-treated hepatocytes in 2 microarray datasets was also compared to locate differentially expressed genes involved in cysteine metabolism. The study demonstrates that increased protein adducts formation and changes in cysteine concentration occur under chronic alcohol condition in liver cells which may increase alcohol-mediated oxidative injury.

  20. Development of heart failure is independent of K+ channel-interacting protein 2 expression

    DEFF Research Database (Denmark)

    Speerschneider, Tobias; Grubb, Søren; Metoska, Artina

    2013-01-01

    Abstract  Abnormal ventricular repolarization in ion channelopathies and heart disease is a major cause of ventricular arrhythmias and sudden cardiac death. K(+) channel-interacting protein 2 (KChIP2) expression is significantly reduced in human heart failure (HF), contributing to a loss of the t......Abstract  Abnormal ventricular repolarization in ion channelopathies and heart disease is a major cause of ventricular arrhythmias and sudden cardiac death. K(+) channel-interacting protein 2 (KChIP2) expression is significantly reduced in human heart failure (HF), contributing to a loss...... of the transient outward K(+) current (Ito). We aim to investigate the possible significance of a changed KChIP2 expression on the development of HF and proarrhythmia. Transverse aortic constrictions (TAC) and sham operations were performed in wild-type (WT) and KChIP2(-/-) mice. Echocardiography was performed...

  1. Intracellular calcium elevation during plateau potentials mediated by extrasynaptic NMDA receptor activation in rat hippocampal CA1 pyramidal neurons is primarily due to calcium entry through voltage-gated calcium channels.

    Science.gov (United States)

    Oda, Yoshiaki; Kodama, Satoshi; Tsuchiya, Sadahiro; Inoue, Masashi; Miyakawa, Hiroyoshi

    2014-05-01

    We reported previously that plateau potentials mediated by extrasynaptic N-methyl-d-aspartate receptors (NMDARs) can be induced either by synaptic stimulation in the presence of glutamate transporter antagonist or by iontophoresis of NMDA in rat hippocampal CA1 pyramidal neurons. To examine whether the plateau potentials are accompanied by an elevation of intracellular Ca2+ and to determine the source of Ca2+ elevation, we performed Ca2+ imaging during the plateau potential. Neurons were loaded with Ca2+ indicator fluo-4, and the plateau potentials were generated either synaptically in the presence of glutamate transporter antagonist or by iontophoretically applying NMDA. We have found that a transient elevation in intracellular Ca2+ accompanies the plateau potential. The synaptically induced plateau potential and the Ca2+ elevation were blocked by 5,7-dichlorokynurenic acid (5,7-dCK), an antagonist for the glycine-binding sites of NMDAR. A mixture of Cd2+ and tetrodotoxin did not block NMDA-induced plateau potentials, but completely abolished the accompanying Ca2+ elevation in both the presence and absence of Mg2+ ions in the bathing solution. The NMDA-induced plateau potential was blocked by further adding 5,7-dCK. Our results show that the NMDAR-mediated plateau potential is accompanied by elevation of intracellular Ca2+ that is primarily caused by the influx of Ca2+ through voltage-gated Ca2+ channels. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  2. G protein-coupled receptors form stable complexes with inwardly rectifying potassium channels and adenylyl cyclase.

    Science.gov (United States)

    Lavine, Natalie; Ethier, Nathalie; Oak, James N; Pei, Lin; Liu, Fang; Trieu, Phan; Rebois, R Victor; Bouvier, Michel; Hebert, Terence E; Van Tol, Hubert H M

    2002-11-29

    A large number of studies have demonstrated co-purification or co-immunoprecipitation of receptors with G proteins. We have begun to look for the presence of effector molecules in these receptor complexes. Co-expression of different channel and receptor permutations in COS-7 and HEK 293 cells in combination with co-immunoprecipitation experiments established that the dopamine D(2) and D(4), and beta(2)-adrenergic receptors (beta(2)-AR) form stable complexes with Kir3 channels. The D(4)/Kir3 and D(2) receptor/Kir3 interaction does not occur when the channel and receptor are expressed separately and mixed prior to immunoprecipitation, indicating that the interaction is not an artifact of the experimental protocol and reflects a biosynthetic event. The observed complexes are stable in that they are not disrupted by receptor activation or modulation of G protein alpha subunit function. However, using a peptide that binds Gbetagamma (betaARKct), we show that Gbetagamma is critical for dopamine receptor-Kir3 complex formation, but not for maintenance of the complex. We also provide evidence that Kir3 channels and another effector, adenylyl cyclase, are stably associated with the beta(2)-adrenergic receptor and can be co-immunoprecipitated by anti-receptor antibodies. Using bioluminescence resonance energy transfer, we have shown that in living cells under physiological conditions, beta(2)AR interacts directly with Kir3.1/3.4 and Kir3.1/3.2c heterotetramers as well as with adenylyl cyclase. All of these interactions are stable in the presence of receptor agonists, suggesting that these signaling complexes persist during signal transduction. In addition, we provide evidence that the receptor-effector complexes are also found in vivo. The observation that several G protein-coupled receptors form stable complexes with their effectors suggests that this arrangement might be a general feature of G protein-coupled signal transduction.

  3. Involvement of intracellular transport in TREK-1c current run-up in 293T cells.

    Science.gov (United States)

    Andharia, Naaz; Joseph, Ancy; Hayashi, Mikio; Okada, Masayoshi; Matsuda, Hiroko

    2017-05-04

    The TREK-1 channel, the TWIK-1-related potassium (K + ) channel, is a member of a family of 2-pore-domain K + (K2P) channels, through which background or leak K + currents occur. An interesting feature of the TREK-1 channel is the run-up of current: i.e. the current through TREK-1 channels spontaneously increases within several minutes of the formation of the whole-cell configuration. To investigate whether intracellular transport is involved in the run-up, we established 293T cell lines stably expressing the TREK-1c channel (K2P2.1) and examined the effects of inhibitors of membrane protein transport, N-methylmaleimide (NEM), brefeldin-A, and an endocytosis inhibitor, pitstop2, on the run-up. The results showing that NEM and brefeldin-A inhibited and pitstop2 facilitated the run-up suggest the involvement of intracellular protein transport. Correspondingly, in cells stably expressing the mCherry-TREK-1 fusion protein, NEM decreased and pitstop2 increased the cell surface localization of the fusion protein. Furthermore, the run-up was inhibited by the intracellular application of a peptide of the C-terminal fragment TREK335-360, corresponding to the interaction site with microtubule-associated protein 2 (Mtap2). This peptide also inhibited the co-immunoprecipitation of Mtap2 with anti-mCherry antibody. The extracellular application of an ezrin inhibitor (NSC668394) also suppressed the run-up and surface localization of the fusion protein. The co-application of these inhibitors abolished the TREK-1c current, suggesting that the additive effects of ezrin and Mtap2 enhance the surface expression of TREK-1c channels and the run-up. These findings clearly showed the involvement of intracellular transport in TREK-1c current run-up and its mechanism.

  4. A Mutation in the Intracellular Loop III/IV of Mosquito Sodium Channel Synergizes the Effect of Mutations in Helix IIS6 on Pyrethroid Resistance

    Science.gov (United States)

    Wang, Lingxin; Nomura, Yoshiko; Du, Yuzhe; Liu, Nannan; Zhorov, Boris S.

    2015-01-01

    Activation and inactivation of voltage-gated sodium channels are critical for proper electrical signaling in excitable cells. Pyrethroid insecticides promote activation and inhibit inactivation of sodium channels, resulting in prolonged opening of sodium channels. They preferably bind to the open state of the sodium channel by interacting with two distinct receptor sites, pyrethroid receptor sites PyR1 and PyR2, formed by the interfaces of domains II/III and I/II, respectively. Specific mutations in PyR1 or PyR2 confer pyrethroid resistance in various arthropod pests and disease vectors. Recently, a unique mutation, N1575Y, in the cytoplasmic loop linking domains III and IV (LIII/IV) was found to coexist with a PyR2 mutation, L1014F in IIS6, in pyrethroid-resistant populations of Anopheles gambiae. To examine the role of this mutation in pyrethroid resistance, N1575Y alone or N1575Y + L1014F were introduced into an Aedes aegypti sodium channel, AaNav1-1, and the mutants were functionally examined in Xenopus oocytes. N1575Y did not alter AaNav1-1 sensitivity to pyrethroids. However, the N1575Y + L1014F double mutant was more resistant to pyrethroids than the L1014F mutant channel. Further mutational analysis showed that N1575Y could also synergize the effect of L1014S/W, but not L1014G or other pyrethroid-resistant mutations in IS6 or IIS6. Computer modeling predicts that N1575Y allosterically alters PyR2 via a small shift of IIS6. Our findings provide the molecular basis for the coexistence of N1575Y with L1014F in pyrethroid resistance, and suggest an allosteric interaction between IIS6 and LIII/IV in the sodium channel. PMID:25523031

  5. Mechanosensitive channels of Escherichia coli: the MscL gene, protein, and activities

    Science.gov (United States)

    Sukharev, S. I.; Blount, P.; Martinac, B.; Kung, C.

    1997-01-01

    Although mechanosensory responses are ubiquitous and diverse, the molecular bases of mechanosensation in most cases remain mysterious MscL, a mechanosensitive channel of large conductance of Escherichia coli and its bacterial homologues are the first and currently only channel molecules shown to directly sense mechanical stretch of the membrane. In response to the tension conveyed via the lipid bilayer, MscL increases its open probability by several orders of magnitude. In the present review we describe the identification, cloning, and first sets of biophysical and structural data on this simplest mechanosensory molecule. We discovered a 2.5-ns mechanosensitive conductance in giant E. coli spheroplasts. Using chromatographies to enrich the target and patch clamp to assay the channel activity in liposome-reconstituted fractions, we identified the MscL protein and cloned the mscL gene. MscL comprises 136 amino acid residues (15 kDa), with two highly hydrophobic regions, and resides in the inner membrane of the bacterium. PhoA-fusion experiments indicate that the protein spans the membrane twice with both termini in the cytoplasm. Spectroscopic techniques show that it is highly helical. Expression of MscL tandems and covalent cross-linking suggest that the active channel complex is a homo-hexamer. We have identified several residues, which when deleted or substituted, affect channel kinetics or mechanosensitivity. Although unique when discovered, highly conserved MscL homologues in both gram-negative and gram-positive bacteria have been found, suggesting their ubiquitous importance among bacteria.

  6. Stage and cell-specific expression and intracellular localization of the small heat shock protein Hsp27 during oogenesis and spermatogenesis in the Mediterranean fruit fly, Ceratitis capitata.

    Science.gov (United States)

    Economou, Katerina; Kotsiliti, Elena; Mintzas, Anastassios C

    2017-01-01

    The cell-specific expression and intracellular distribution of the small heat protein Hsp27 was investigated in the ovaries and testes of the Mediterranean fruit fly, Ceratitis capitata (medfly), under both normal and heat shock conditions. For this study, a gfp-hsp27 strain was used to detect the chimeric protein by confocal microscopy. In unstressed ovaries, the protein was expressed throughout egg development in a stage and cell-specific pattern. In germarium, the protein was detected in the cytoplasm of the somatic cells in both unstressed and heat-shocked ovaries. In the early stages of oogenesis of unstressed ovaries, the protein was mainly located in the perinuclear region of the germ cells and in the cytoplasm of the follicle cells, while in later stages (9-10) it was distributed in the cytoplasm of the germ cells. In late stages (12-14), the protein changed localization pattern and was exclusively associated with the nuclei of the somatic cells. In heat shocked ovaries, the protein was mainly located in the nuclei of the somatic cells throughout egg chamber's development. In unstressed testes, the chimeric protein was detected in the nuclei of primary spermatocytes and in the filamentous structures of spermatid bundles, called actin cones. Interestingly, after a heat shock, the protein presented the same cell-specific localization pattern as in unstressed testes. Furthermore, the protein was also detected in the nuclei of the epithelial cells of the deferent duct, the accessory glands and the ejaculatory bulb. Our data suggest that medfly Hsp27 may have cell-specific functions, especially in the nucleus. Moreover, the association of this protein to actin cones during spermatid individualization, suggests a possible role of the protein in the formation and stabilization of actin cones. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Butyrate increases intracellular calcium levels and enhances growth hormone release from rat anterior pituitary cells via the G-protein-coupled receptors GPR41 and 43.

    Directory of Open Access Journals (Sweden)

    Maria Consolata Miletta

    Full Text Available Butyrate is a short-chain fatty acid (SCFA closely related to the ketone body ß-hydroxybutyrate (BHB, which is considered to be the major energy substrate during prolonged exercise or starvation. During fasting, serum growth hormone (GH rises concomitantly with the accumulation of BHB and butyrate. Interactions between GH, ketone bodies and SCFA during the metabolic adaptation to fasting have been poorly investigated to date. In this study, we examined the effect of butyrate, an endogenous agonist for the two G-protein-coupled receptors (GPCR, GPR41 and 43, on non-stimulated and GH-releasing hormone (GHRH-stimulated hGH secretion. Furthermore, we investigated the potential role of GPR41 and 43 on the generation of butyrate-induced intracellular Ca2+ signal and its ultimate impact on hGH secretion. To study this, wt-hGH was transfected into a rat pituitary tumour cell line stably expressing the human GHRH receptor. Treatment with butyrate promoted hGH synthesis and improved basal and GHRH-induced hGH-secretion. By acting through GPR41 and 43, butyrate enhanced intracellular free cytosolic Ca2+. Gene-specific silencing of these receptors led to a partial inhibition of the butyrate-induced intracellular Ca2+ rise resulting in a decrease of hGH secretion. This study suggests that butyrate is a metabolic intermediary, which contributes to the secretion and, therefore, to the metabolic actions of GH during fasting.

  8. Butyrate increases intracellular calcium levels and enhances growth hormone release from rat anterior pituitary cells via the G-protein-coupled receptors GPR41 and 43.

    Science.gov (United States)

    Miletta, Maria Consolata; Petkovic, Vibor; Eblé, Andrée; Ammann, Roland A; Flück, Christa E; Mullis, Primus-E

    2014-01-01

    Butyrate is a short-chain fatty acid (SCFA) closely related to the ketone body ß-hydroxybutyrate (BHB), which is considered to be the major energy substrate during prolonged exercise or starvation. During fasting, serum growth hormone (GH) rises concomitantly with the accumulation of BHB and butyrate. Interactions between GH, ketone bodies and SCFA during the metabolic adaptation to fasting have been poorly investigated to date. In this study, we examined the effect of butyrate, an endogenous agonist for the two G-protein-coupled receptors (GPCR), GPR41 and 43, on non-stimulated and GH-releasing hormone (GHRH)-stimulated hGH secretion. Furthermore, we investigated the potential role of GPR41 and 43 on the generation of butyrate-induced intracellular Ca2+ signal and its ultimate impact on hGH secretion. To study this, wt-hGH was transfected into a rat pituitary tumour cell line stably expressing the human GHRH receptor. Treatment with butyrate promoted hGH synthesis and improved basal and GHRH-induced hGH-secretion. By acting through GPR41 and 43, butyrate enhanced intracellular free cytosolic Ca2+. Gene-specific silencing of these receptors led to a partial inhibition of the butyrate-induced intracellular Ca2+ rise resulting in a decrease of hGH secretion. This study suggests that butyrate is a metabolic intermediary, which contributes to the secretion and, therefore, to the metabolic actions of GH during fasting.

  9. G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na/sup +/ channel interaction

    Energy Technology Data Exchange (ETDEWEB)

    Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

    1988-01-12

    The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na/sup +/ channels is a coupled event mediated by guanine nucleotide binding protein(s) (G-protein(s)). These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of (/sup 3/H) acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of (/sup 3/H)batrachotoxin to Na/sup +/ channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced /sup 22/Na/sup +/ uptake in the presence and absence of tetrodotoxin, which blocks Na/sup +/ channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na/sup +/channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na/sup +/ channel-is such that at resting potential the muscarinic receptor induces opening of Na/sup +/ channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues.

  10. G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na+ channel interaction

    International Nuclear Information System (INIS)

    Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

    1988-01-01

    The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na + channels is a coupled event mediated by guanine nucleotide binding protein(s) [G-protein(s)]. These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of [ 3 H] acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of [ 3 H]batrachotoxin to Na + channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced 22 Na + uptake in the presence and absence of tetrodotoxin, which blocks Na + channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na + channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na + channel-is such that at resting potential the muscarinic receptor induces opening of Na + channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues

  11. The intracellular Scots pine shoot symbiont Methylobacterium extorquens DSM13060 aggregates around the host nucleus and encodes eukaryote-like proteins.

    Science.gov (United States)

    Koskimäki, Janne J; Pirttilä, Anna Maria; Ihantola, Emmi-Leena; Halonen, Outi; Frank, A Carolin

    2015-03-24

    Endophytes are microbes that inhabit plant tissues without any apparent signs of infection, often fundamentally altering plant phenotypes. While endophytes are typically studied in plant roots, where they colonize the apoplast or dead cells, Methylobacterium extorquens strain DSM13060 is a facultatively intracellular symbiont of the meristematic cells of Scots pine (Pinus sylvestris L.) shoot tips. The bacterium promotes host growth and development without the production of known plant growth-stimulating factors. Our objective was to examine intracellular colonization by M. extorquens DSM13060 of Scots pine and sequence its genome to identify novel molecular mechanisms potentially involved in intracellular colonization and plant growth promotion. Reporter construct analysis of known growth promotion genes demonstrated that these were only weakly active inside the plant or not expressed at all. We found that bacterial cells accumulate near the nucleus in intact, living pine cells, pointing to host nuclear processes as the target of the symbiont's activity. Genome analysis identified a set of eukaryote-like functions that are common as effectors in intracellular bacterial pathogens, supporting the notion of intracellular bacterial activity. These include ankyrin repeats, transcription factors, and host-defense silencing functions and may be secreted by a recently imported type IV secretion system. Potential factors involved in host growth include three copies of phospholipase A2, an enzyme that is rare in bacteria but implicated in a range of plant cellular processes, and proteins putatively involved in gibberellin biosynthesis. Our results describe a novel endophytic niche and create a foundation for postgenomic studies of a symbiosis with potential applications in forestry and agriculture. All multicellular eukaryotes host communities of essential microbes, but most of these interactions are still poorly understood. In plants, bacterial endophytes are found inside

  12. Melatonin-Mediated Intracellular Insulin during 2-Deoxy-d-glucose Treatment Is Reduced through Autophagy and EDC3 Protein in Insulinoma INS-1E Cells

    Directory of Open Access Journals (Sweden)

    Han Sung Kim

    2016-01-01

    Full Text Available 2-DG triggers glucose deprivation without altering other nutrients or metabolic pathways and then activates autophagy via activation of AMPK and endoplasmic reticulum (ER stress. We investigated whether 2-DG reduced intracellular insulin increased by melatonin via autophagy/EDC3 in insulinoma INS-1E cells. p-AMPK and GRP78/BiP level were significantly increased by 2-DG in the presence/absence of melatonin, but IRE1α level was reduced in 2-DG treatment. Levels of p85α, p110, p-Akt (Ser473, Thr308, and p-mTOR (Ser2481 were also significantly reduced by 2-DG in the presence/absence of melatonin. Mn-SOD increased with 2-DG plus melatonin compared to groups treated with/without melatonin alone. Bcl-2 was decreased and Bax increased with 2-DG plus melatonin. LC3II level increased with 2-DG treatment in the presence/absence of melatonin. Intracellular insulin production increased in melatonin plus 2-DG but reduced in treatment with 2-DG with/without melatonin. EDC3 was increased by 2-DG in the presence/absence of melatonin. Rapamycin, an mTOR inhibitor, increased GRP78/BiP and EDC3 levels in a dose-dependent manner and subsequently resulted in a decrease in intracellular production of insulin. These results suggest that melatonin-mediated insulin synthesis during 2-DG treatment involves autophagy and EDC3 protein in rat insulinoma INS-1E cells and subsequently results in a decrease in intracellular production of insulin.

  13. Expression of orphan G-protein coupled receptor GPR174 in CHO cells induced morphological changes and proliferation delay via increasing intracellular cAMP

    Energy Technology Data Exchange (ETDEWEB)

    Sugita, Kazuya; Yamamura, Chiaki; Tabata, Ken-ichi [Laboratory of Pharmacoinformatics, Graduate School of Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); Fujita, Norihisa, E-mail: nori@ph.ritsumei.ac.jp [Laboratory of Pharmacoinformatics, Graduate School of Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); School of Pharmacy, Ristumeikan University, Kusatsu, Shiga 525-8577 (Japan)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Expression of GPR174 in CHO cells induces morphological changes and proliferation delay. Black-Right-Pointing-Pointer These are due to increase in intracellular cAMP concentration. Black-Right-Pointing-Pointer Lysophosphatidylserine was identified to stimulate GPR174 leading to activate ACase. Black-Right-Pointing-Pointer The potencies of fatty acid moiety on LysoPS were oleoyl Greater-Than-Or-Slanted-Equal-To stearoyl > palmitoyl. Black-Right-Pointing-Pointer We propose that GPR174 is a lysophosphatidylserine receptor. -- Abstract: We established cell lines that stably express orphan GPCR GPR174 using CHO cells, and studied physiological and pharmacological features of the receptor. GPR174-expressing cells showed cell-cell adhesion with localization of actin filaments to cell membrane, and revealed significant delay of cell proliferation. Since the morphological changes of GPR174-cells were very similar to mock CHO cells treated with cholera toxin, we measured the concentration of intracellular cAMP. The results showed the concentration was significantly elevated in GPR174-cells. By measuring intracellular cAMP concentration in GPR174-cells, we screened lipids and nucleotides to identify ligands for GPR174. We found that lysophosphatidylserine (LysoPS) stimulated increase in intracellular cAMP in a dose-dependent manner. Moreover, phosphorylation of Erk was elevated by LysoPS in GPR174 cells. These LysoPS responses were inhibited by NF449, an inhibitor of G{alpha}{sub s} protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with G{alpha}{sub s}, and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation.

  14. Mouse taste cells with G protein-coupled taste receptors lack voltage-gated calcium channels and SNAP-25

    Directory of Open Access Journals (Sweden)

    Medler Kathryn F

    2006-03-01

    Full Text Available Abstract Background Taste receptor cells are responsible for transducing chemical stimuli from the environment and relaying information to the nervous system. Bitter, sweet and umami stimuli utilize G-protein coupled receptors which activate the phospholipase C (PLC signaling pathway in Type II taste cells. However, it is not known how these cells communicate with the nervous system. Previous studies have shown that the subset of taste cells that expresses the T2R bitter receptors lack voltage-gated Ca2+ channels, which are normally required for synaptic transmission at conventional synapses. Here we use two lines of transgenic mice expressing green fluorescent protein (GFP from two taste-specific promoters to examine Ca2+ signaling in subsets of Type II cells: T1R3-GFP mice were used to identify sweet- and umami-sensitive taste cells, while TRPM5-GFP mice were used to identify all cells that utilize the PLC signaling pathway for transduction. Voltage-gated Ca2+ currents were assessed with Ca2+ imaging and whole cell recording, while immunocytochemistry was used to detect expression of SNAP-25, a presynaptic SNARE protein that is associated with conventional synapses in taste cells. Results Depolarization with high K+ resulted in an increase in intracellular Ca2+ in a small subset of non-GFP labeled cells of both transgenic mouse lines. In contrast, no depolarization-evoked Ca2+ responses were observed in GFP-expressing taste cells of either genotype, but GFP-labeled cells responded to the PLC activator m-3M3FBS, suggesting that these cells were viable. Whole cell recording indicated that the GFP-labeled cells of both genotypes had small voltage-dependent Na+ and K+ currents, but no evidence of Ca2+ currents. A subset of non-GFP labeled taste cells exhibited large voltage-dependent Na+ and K+ currents and a high threshold voltage-gated Ca2+ current. Immunocytochemistry indicated that SNAP-25 was expressed in a separate population of taste cells

  15. Clinical spectrum and diagnostic value of antibodies against the potassium channel-related protein complex☆

    Science.gov (United States)

    Montojo, M.T.; Petit-Pedrol, M.; Graus, F.; Dalmau, J.

    2016-01-01

    Introduction Antibodies against a protein complex that includes voltage-gated potassium channels (VGKC) have been reported in patients with limbic encephalitis, peripheral nerve hyperexcitability, Morvan's syndrome, and a large variety of neurological syndromes. Review summary In this article, a review is presented of the syndromes associated with antibodies against VGKC-related proteins and the main antigens of this protein complex, the proteins LGI1 (leucine rich glioma inactivated protein 1) and Caspr2 (contactin-associated protein-like 2). The conceptual problems and clinical implications of the description of antibodies against VGKC-related proteins other than LGI1 and Caspr2 are also discussed. Although initial studies indicated the occurrence of antibodies against VGKC, recent investigations have shown that the main antigens are a neuronal secreted protein known as LGI1 which modulates synaptic excitability, and a protein called Caspr2 located on the cell surface and processes of neurons of different brain regions, and at the juxtaparanodal region of myelinated axons. While antibodies against LGI1 preferentially associate with classical limbic encephalitis, antibodies against Caspr2 associate with a wider spectrum of symptoms, including Morvan's syndrome, peripheral nerve hyperexcitability or neuromyotonia, and limbic or more extensive encephalitis. In addition there are reports of patients with antibodies against VGKC-related proteins that are different from LGI1 or Caspr2. In these cases, the identity and location of the antigens are unknown, the syndrome association is not specific, and the response to treatment uncertain. Conclusions The discovery of antigens such as LGI1 and Caspr2 has resulted in a clinical and molecular definition of the broad group of diseases previously attributed to antibodies against VGKC. Considering the literature that describes the presence of antibodies against VGKC other than LGI1 and Caspr2 proteins, we propose a practical

  16. Do TRPC channels support working memory? Comparing modulations of TRPC channels and working memory through G-protein coupled receptors and neuromodulators.

    Science.gov (United States)

    Reboreda, Antonio; Theissen, Frederik M; Valero-Aracama, Maria; Arboit, Alberto; Corbu, Mihaela A; Yoshida, Motoharu

    2018-03-01

    Working memory is a crucial ability we use in daily life. However, the cellular mechanisms supporting working memory still remain largely unclear. A key component of working memory is persistent neural firing which is believed to serve short-term (hundreds of milliseconds up to tens of seconds) maintenance of necessary information. In this review, we will focus on the role of transient receptor potential canonical (TRPC) channels as a mechanism underlying persistent firing. Many years of in vitro work have been suggesting a crucial role of TRPC channels in working memory and temporal association tasks. If TRPC channels are indeed a central mechanism for working memory, manipulations which impair or facilitate working memory should have a similar effect on TRPC channel modulation. However, modulations of working memory and TRPC channels were never systematically compared, and it remains unanswered whether TRPC channels indeed contribute to working memory in vivo or not. In this article, we review the effects of G-protein coupled receptors (GPCR) and neuromodulators, including acetylcholine, noradrenalin, serotonin and dopamine, on working memory and TRPC channels. Based on comparisons, we argue that GPCR and downstream signaling pathways that activate TRPC, generally support working memory, while those that suppress TRPC channels impair it. However, depending on the channel types, areas, and systems tested, this is not the case in all studies. Further work to clarify involvement of specific TRPC channels in working memory tasks and how they are affected by neuromodulators is still necessary in the future. Copyright © 2018. Published by Elsevier B.V.

  17. RING finger protein 121 facilitates the degradation and membrane localization of voltage-gated sodium channels

    Science.gov (United States)

    Ogino, Kazutoyo; Low, Sean E.; Yamada, Kenta; Saint-Amant, Louis; Zhou, Weibin; Muto, Akira; Asakawa, Kazuhide; Nakai, Junichi; Kawakami, Koichi; Kuwada, John Y.; Hirata, Hiromi

    2015-01-01

    Following their synthesis in the endoplasmic reticulum (ER), voltage-gated sodium channels (NaV) are transported to the membranes of excitable cells, where they often cluster, such as at the axon initial segment of neurons. Although the mechanisms by which NaV channels form and maintain clusters have been extensively examined, the processes that govern their transport and degradation have received less attention. Our entry into the study of these processes began with the isolation of a new allele of the zebrafish mutant alligator, which we found to be caused by mutations in the gene encoding really interesting new gene (RING) finger protein 121 (RNF121), an E3-ubiquitin ligase present in the ER and cis-Golgi compartments. Here we demonstrate that RNF121 facilitates two opposing fates of NaV channels: (i) ubiquitin-mediated proteasome degradation and (ii) membrane localization when coexpressed with auxiliary NaVβ subunits. Collectively, these results indicate that RNF121 participates in the quality control of NaV channels during their synthesis and subsequent transport to the membrane. PMID:25691753

  18. Role of protein sulfation in vasodilation induced by minoxidil sulfate, a K+ channel opener

    Energy Technology Data Exchange (ETDEWEB)

    Meisheri, K.D.; Oleynek, J.J.; Puddington, L. (Cardiovascular Diseases Research, Upjohn Laboratories, Upjohn Company, Kalamazoo, MI (United States))

    1991-09-01

    Evidence from contractile, radioisotope ion flux and electrophysiological studies suggest that minoxidil sulfate (MNXS) acts as a K+ channel opener in vascular smooth muscle. This study was designed to examine possible biochemical mechanisms by which MNXS exerts such an effect. Experiments performed in the isolated rabbit mesenteric artery (RMA) showed that MNXS, 5 microM, but not the parent compound minoxidil, was a potent vasodilator. Whereas the relaxant effects of an another K+ channel opener vasodilator, BRL-34915 (cromakalim), were removed by washing with physiological saline solution, the effects of MNXS persisted after repeated washout attempts. Furthermore, after an initial exposure of segments of intact RMA to (35S) MNXS, greater than 30% of the radiolabel was retained 2 hr after removal of the drug. In contrast, retention of radiolabel was not detected with either (3H)MNXS (label on the piperidine ring of MNXS) or (3H)minoxidil (each less than 3% after a 2-hr washout). These data suggested that the sulfate moiety from MNXS was closely associated with the vascular tissue. To determine if proteins were the acceptors of sulfate from MNXS, intact RMAs were incubated with (35S)MNXS, and then 35S-labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by fluorography. Preferential labeling of a 116 kD protein was detected by 2 and 5 min of treatment. A 43 kD protein (resembling actin) also showed significant labeling. A similar profile of 35S-labeled proteins was observed in (35S) MNXS-treated A7r5 rat aortic smooth muscle cells, suggesting that the majority of proteins labeled by (35S)MNXS in intact RMA were components of smooth muscle cells.

  19. Expression of Transient Receptor Potential Canonical Channel Proteins in Human Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Qi ZHANG

    2010-06-01

    Full Text Available Background and objective Transient receptor potential canonical (TRPC proteins, a group of Ca2+ permeable nonselective cation channels, are thought to constitute store-operated calcium channels (SOCC and mediate store-operated calcium entry (SOCE in various cell types. Members of TRPC have been found to be involved in abnormal proliferation, differentiation, and growth of cancer cells. The aim of this study is to detect the mRNA and protein expression of TRPC in non-small cell lung cancer (NSCLC. Methods Real-time quantitative PCR was performed to screen the expression of TRPC mRNA in NSCLC tissue. Protein expression of TRPC was detected by Western blot. Results Among the seven family members of TRPC so far identified (TRPC1-7, we detected the expression of TRPC1, TRPC3, TRPC4, TRPC6 mRNA in 24 cases of NSCLC tissue; TRPC2, TRPC5 and TRPC7 mRNA were not detectable. The relative abundance of the expressed TRPC was TRPC1≈TRPC6>TRPC3>TRPC4. Western blot confirmed the protein expression of TRPC1, TRPC3, TRPC4 and TRPC6 in NSCLC tissue. Conclusion Out of the seven members of TRPC, we found TRPC1, TRPC3, TRPC4, TRPC6 mRNA and protein were selectively expressed in human NSCLC tissue. This study could provide a basis for future exploration of the individual role of these TRPC proteins in mediating SOCE and in the progression of lung cancer.

  20. Effects of protein-protein interactions and ligand binding on the ion permeation in KCNQ1 potassium channel.

    Science.gov (United States)

    Jalily Hasani, Horia; Ganesan, Aravindhan; Ahmed, Marawan; Barakat, Khaled H

    2018-01-01

    The voltage-gated KCNQ1 potassium ion channel interacts with the type I transmembrane protein minK (KCNE1) to generate the slow delayed rectifier (IKs) current in the heart. Mutations in these transmembrane proteins have been linked with several heart-related issues, including long QT syndromes (LQTS), congenital atrial fibrillation, and short QT syndrome. Off-target interactions of several drugs with that of KCNQ1/KCNE1 ion channel complex have been known to cause fatal cardiac irregularities. Thus, KCNQ1/KCNE1 remains an important avenue for drug-design and discovery research. In this work, we present the structural and mechanistic details of potassium ion permeation through an open KCNQ1 structural model using the combined molecular dynamics and steered molecular dynamics simulations. We discuss the processes and key residues involved in the permeation of a potassium ion through the KCNQ1 ion channel, and how the ion permeation is affected by (i) the KCNQ1-KCNE1 interactions and (ii) the binding of chromanol 293B ligand and its derivatives into the complex. The results reveal that interactions between KCNQ1 with KCNE1 causes a pore constriction in the former, which in-turn forms small energetic barriers in the ion-permeation pathway. These findings correlate with the previous experimental reports that interactions of KCNE1 dramatically slows the activation of KCNQ1. Upon ligand-binding onto the complex, the energy-barriers along ion permeation path are more pronounced, as expected, therefore, requiring higher force in our steered-MD simulations. Nevertheless, pulling the ion when a weak blocker is bound to the channel does not necessitate high force in SMD. This indicates that our SMD simulations have been able to discern between strong and week blockers and reveal their influence on potassium ion permeation. The findings presented here will have some implications in understanding the potential off-target interactions of the drugs with the KCNQ1/KCNE1 channel

  1. Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis

    Science.gov (United States)

    Suh, Jin Sook; Lee, Jue Yeon; Choi, Yoon Jung; You, Hyung Keun; Hong, Seong-Doo; Chung, Chong Pyoung; Park, Yoon Jeong

    2014-01-01

    Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP), and a transcriptional coactivator with a PDZ-binding motif (TAZ) protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC) differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in alginate gel for the purpose of localization and controlled release. The LMWP-TAZ fusion protein-loaded alginate gel matrix significantly increased bone formation in rabbit calvarial defects compared with alginate gel matrix mixed with free TAZ protein. The protein transduction of TAZ fused with cell-penetrating LMWP peptide was able selectively to stimulate osteogenesis in vitro and in vivo. Taken together, this fusion protein-transduction technology for osteogenic protein can thus be applied in combination with biomaterials for tissue regeneration and controlled release for tissue

  2. Channels Formed by Botulinum, Tetanus, and Diphtheria Toxins in Planar Lipid Bilayers: Relevance to Translocation of Proteins across Membranes

    Science.gov (United States)

    Hoch, David H.; Romero-Mira, Miryam; Ehrlich, Barbara E.; Finkelstein, Alan; Dasgupta, Bibhuti R.; Simpson, Lance L.

    1985-03-01

    The heavy chains of both botulinum neurotoxin type B and tetanus toxin form channels in planar bilayer membranes. These channels have pH-dependent and voltage-dependent properties that are remarkably similar to those previously described for diphtheria toxin. Selectivity experiments with anions and cations show that the channels formed by the heavy chains of all three toxins are large; thus, these channels could serve as ``tunnel proteins'' for translocation of active peptide fragments. These findings support the hypothesis that the active fragments of botulinum neurotoxin and tetanus toxin, like that of diphtheria toxin, are translocated across the membranes of acidic vesicles.

  3. Taste receptors and gustatory associated G proteins in channel catfish, Ictalurus punctatus.

    Science.gov (United States)

    Gao, Sen; Liu, Shikai; Yao, Jun; Zhou, Tao; Li, Ning; Li, Qi; Dunham, Rex; Liu, Zhanjiang

    2017-03-01

    Taste sensation plays a pivotal role in nutrient identification and acquisition. This is particularly true for channel catfish (Ictalurus punctatus) that live in turbid waters with limited visibility. This biological process is mainly mediated by taste receptors expressed in taste buds that are distributed in several organs and tissues, including the barbels and skin. In the present study, we identified a complete repertoire of taste receptor and gustatory associated G protein genes in the channel catfish genome. A total of eight taste receptor genes were identified, including five type I and three type II taste receptor genes. Their genomic locations, phylogenetic relations, orthologies and expression were determined. Phylogenetic and collinear analyses provided understanding of the evolution dynamics of this gene family. Furthermore, the motif and dN/dS analyses indicated that selection pressures of different degrees were imposed on these receptors. Additionally, four genes of gustatory associated G proteins were also identified. It was indicated that expression patterns of catfish taste receptors and gustatory associated G proteins across organs mirror the distribution of taste buds across organs. Finally, the expression comparison between catfish and zebrafish organs provided evidence of potential roles of catfish skin and gill involved in taste sensation. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Golgi anti-apoptotic protein: a tale of camels, calcium, channels and cancer.

    Science.gov (United States)

    Carrara, Guia; Parsons, Maddy; Saraiva, Nuno; Smith, Geoffrey L

    2017-05-01

    Golgi anti-apoptotic protein (GAAP), also known as transmembrane Bax inhibitor-1 motif-containing 4 (TMBIM4) or Lifeguard 4 (Lfg4), shares remarkable amino acid conservation with orthologues throughout eukaryotes, prokaryotes and some orthopoxviruses, suggesting a highly conserved function. GAAPs regulate Ca 2+ levels and fluxes from the Golgi and endoplasmic reticulum, confer resistance to a broad range of apoptotic stimuli, promote cell adhesion and migration via the activation of store-operated Ca 2+ entry, are essential for the viability of human cells, and affect orthopoxvirus virulence. GAAPs are oligomeric, multi-transmembrane proteins that are resident in Golgi membranes and form cation-selective ion channels that may explain the multiple functions of these proteins. Residues contributing to the ion-conducting pore have been defined and provide the first clues about the mechanistic link between these very different functions of GAAP. Although GAAPs are naturally oligomeric, they can also function as monomers, a feature that distinguishes them from other virus-encoded ion channels that must oligomerize for function. This review summarizes the known functions of GAAPs and discusses their potential importance in disease. © 2017 The Authors.

  5. Aquaporin-11: A channel protein lacking apparent transport function expressed in brain

    Directory of Open Access Journals (Sweden)

    Tsunenari Takashi

    2006-05-01

    Full Text Available Abstract Background The aquaporins are a family of integral membrane proteins composed of two subfamilies: the orthodox aquaporins, which transport only water, and the aquaglyceroporins, which transport glycerol, urea, or other small solutes. Two recently described aquaporins, numbers 11 and 12, appear to be more distantly related to the other mammalian aquaporins and aquaglyceroporins. Results We report on the characterization of Aquaporin-11 (AQP11. AQP11 RNA and protein is found in multiple rat tissues, including kidney, liver, testes and brain. AQP11 has a unique distribution in brain, appearing in Purkinje cell dendrites, hippocampal neurons of CA1 and CA2, and cerebral cortical neurons. Immunofluorescent staining of Purkinje cells indicates that AQP11 is intracellular. Unlike other aquaporins, Xenopus oocytes expressing AQP11 in the plasma membrane failed to transport water, glycerol, urea, or ions. Conclusion AQP11 is functionally distinct from other proteins of the aquaporin superfamily and could represent a new aquaporin subfamily. Further studies are necessary to elucidate the role of AQP11 in the brain.

  6. Comparative Proteomics of Purified Pathogen Vacuoles Correlates Intracellular Replication of Legionella pneumophila with the Small GTPase Ras-related protein 1 (Rap1).

    Science.gov (United States)

    Schmölders, Johanna; Manske, Christian; Otto, Andreas; Hoffmann, Christine; Steiner, Bernhard; Welin, Amanda; Becher, Dörte; Hilbi, Hubert

    2017-04-01

    Legionella pneumophila is an opportunistic bacterial pathogen that causes a severe lung infection termed "Legionnaires' disease." The pathogen replicates in environmental protozoa as well as in macrophages within a unique membrane-bound compartment, the Legionella -containing-vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates ca. 300 "effector proteins" into host cells, where they target distinct host factors. The L. pneumophila "pentuple" mutant (Δpentuple) lacks 5 gene clusters (31% of the effector proteins) and replicates in macrophages but not in Dictyostelium discoideum amoeba. To elucidate the host factors defining a replication-permissive compartment, we compare here the proteomes of intact LCVs isolated from D. discoideum or macrophages infected with Δpentuple or the parental strain Lp02. This analysis revealed that the majority of host proteins are shared in D. discoideum or macrophage LCVs containing the mutant or the parental strain, respectively, whereas some proteins preferentially localize to distinct LCVs. The small GTPase Rap1 was identified on D. discoideum LCVs containing strain Lp02 but not the Δpentuple mutant and on macrophage LCVs containing either strain. The localization pattern of active Rap1 on D. discoideum or macrophage LCVs was confirmed by fluorescence microscopy and imaging flow cytometry, and the depletion of Rap1 by RNA interference significantly reduced the intracellular growth of L. pneumophila Thus, comparative proteomics identified Rap1 as a novel LCV host component implicated in intracellular replication of L. pneumophila . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Control of endothelial cell tube formation by Notch ligand intracellular domain interactions with activator protein 1 (AP-1)

    DEFF Research Database (Denmark)

    Forghany, Zary; Robertson, Francesca; Lundby, Alicia

    2018-01-01

    work further demonstrated that JUN strongly stimulates endothelial cell tube formation and that DLL4 constrains this process. These results raise the possibility that Notch/DLL4 signaling is bi-directional and suggest that the DLL4 ICD could represent a point of cross-talk between Notch and receptor...... characterized, accumulating evidence points to the existence of multiple, less well-defined, layers of regulation. In this study, we investigated the function of the intracellular domain (ICD) of the Notch ligand Delta-like 4 (DLL4). We provide evidence that the DLL4 ICD is required for normal DLL4 subcellular......Notch signaling is a ubiquitous signal transduction pathway found in most if not all metazoan cell types characterized to date. It is indispensable for cell differentiation as well as tissue growth, tissue remodelling and apoptosis. Although the canonical Notch signaling pathway is well...

  8. Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis

    Directory of Open Access Journals (Sweden)

    Suh JS

    2014-03-01

    Full Text Available Jin Sook Suh,1,* Jue Yeon Lee,2,* Yoon Jung Choi,1 Hyung Keun You,3 Seong-Doo Hong,4 Chong Pyoung Chung,2 Yoon Jeong Park1,2 1Dental Regenerative Biotechnology, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, 2Central Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC, Seoul, 3Department of Periodontology, College of Dentistry, Wonkwang University, Iksan, 4Department of Oral Pathology, School of Dentistry, Seoul National University, Seoul, Republic of Korea *These authors contributed equally to this work Abstract: Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP, and a transcriptional coactivator with a PDZ-binding motif (TAZ protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in

  9. Intracellular route and biological activity of exogenously delivered Rep proteins from the adeno-associated virus type 2

    International Nuclear Information System (INIS)

    Awedikian, Rafi; Francois, Achille; Guilbaud, Mickael; Moullier, Philippe; Salvetti, Anna

    2005-01-01

    The two large Rep proteins, Rep78 and Rep68, from the adeno-associated virus type 2 (AAV-2) are required for AAV-2 DNA replication, site-specific integration, and for the regulation of viral gene expression. The study of their activities is dependent on the ability to deliver these proteins to the cells in a time and dose-dependent manner. We evaluated the ability of a protein transduction domain (PTD) derived from the human immunodeficiency virus 1 (HIV-1) TAT protein to drive the cellular internalization of exogenously delivered PTD-fused Rep68 proteins. This analysis unexpectedly revealed that recombinant Rep68 alone, in the absence of any PTD, could be endocytosed by the cells. Rep68 as the chimeric TAT-Rep68 proteins were internalized through endocytosis in clathrin-coated vesicles and retained in late endosomes/lysosomes with no detectable nuclear localization. In the presence of adenovirus, the Rep proteins could translocate into the nucleus where they displayed a biological activity. These findings support recent reports on the mechanism of entry of TAT-fused proteins and also revealed a new property of Rep68

  10. Phylogenetic conservation of protein-lipid motifs in pentameric ligand-gated ion channels.

    Science.gov (United States)

    Barrantes, Francisco J

    2015-09-01

    Using the crosstalk between the nicotinic acetylcholine receptor (nAChR) and its lipid microenvironment as a paradigm, this short overview analyzes the occurrence of structural motifs which appear not only to be conserved within the nAChR family and contemporary eukaryotic members of the pentameric ligand-gated ion channel (pLGIC) superfamily, but also extend to prokaryotic homologues found in bacteria. The evolutionarily conserved design is manifested in: 1) the concentric three-ring architecture of the transmembrane region, 2) the occurrence in this region of distinct lipid consensus motifs in prokaryotic and eukaryotic pLGIC and 3) the key participation of the outer TM4 ring in conveying the influence of the lipid membrane environment to the middle TM1-TM3 ring and this, in turn, to the inner TM2 channel-lining ring, which determines the ion selectivity of the channel. The preservation of these constant structural-functional features throughout such a long phylogenetic span likely points to the successful gain-of-function conferred by their early acquisition. This article is part of a Special Issue entitled: Lipid-protein interactions. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Protein structure and ionic selectivity in calcium channels: selectivity filter size, not shape, matters.

    Science.gov (United States)

    Malasics, Attila; Gillespie, Dirk; Nonner, Wolfgang; Henderson, Douglas; Eisenberg, Bob; Boda, Dezso

    2009-12-01

    Calcium channels have highly charged selectivity filters (4 COO(-) groups) that attract cations in to balance this charge and minimize free energy, forcing the cations (Na(+) and Ca(2+)) to compete for space in the filter. A reduced model was developed to better understand the mechanism of ion selectivity in calcium channels. The charge/space competition (CSC) mechanism implies that Ca(2+) is more efficient in balancing the charge of the filter because it provides twice the charge as Na(+) while occupying the same space. The CSC mechanism further implies that the main determinant of Ca(2+) versus Na(+) selectivity is the density of charged particles in the selectivity filter, i.e., the volume of the filter (after fixing the number of charged groups in the filter). In this paper we test this hypothesis by changing filter length and/or radius (shape) of the cylindrical selectivity filter of our reduced model. We show that varying volume and shape together has substantially stronger effects than varying shape alone with volume fixed. Our simulations show the importance of depletion zones of ions in determining channel conductance calculated with the integrated Nernst-Planck equation. We show that confining the protein side chains with soft or hard walls does not influence selectivity.

  12. Highly specific detection of muscarinic M3 receptor, G protein interaction and intracellular trafficking in human detrusor using Proximity Ligation Assay (PLA).

    Science.gov (United States)

    Berndt-Paetz, Mandy; Herbst, Luise; Weimann, Annett; Gonsior, Andreas; Stolzenburg, Jens-Uwe; Neuhaus, Jochen

    2018-03-15

    Muscarinic acetylcholine receptors (mAChRs) regulate a number of important physiological functions. Alteration of mAChR expression or function has been associated in the etiology of several pathologies including functional bladder disorders (e.g bladder pain syndrome/interstitial cystitis - BPS/IC). In a previous study we found specific mAChR expression patterns associated with BPS/IC, while correlation between protein and gene expression was lacking. Posttranslational regulatory mechanisms, e.g. altered intracellular receptor trafficking, could explain those differences. In addition, alternative G protein (GP) coupling could add to the pathophysiology via modulation of muscarinic signaling. In our proof-of-principle study, we addressed these questions in situ. We established PLA in combination with confocal laserscanning microscopy (CLSM) and 3D object reconstruction for highly specific detection and analysis of muscarinic 3 receptors (M3), G protein (GP) coupling and intracellular trafficking in human detrusor samples. Paraffin sections of formalin-fixed bladder tissue (FFPE) of BPS/IC patients receiving transurethral biopsy were examined by Cy3-PLA for M3 expression, coupling of M3 to GPs (G αq/11 , G αs , G αi ) and interaction of M3 with endocytic regulator proteins. Membranes were labeled with wheat germ agglutinin-Alexa Fluor ® 488, nuclei were stained with DAPI. Object density and co-localization were analyzed in 3D-reconstruction of high resolution confocal z-stacks. Confocal image stack processing resulted in well demarcated objects. Calculated receptor densities correlated significantly with existing confocal expression data, while significantly improved specificity of M3 detection by PLA was verified using bladder tissue samples from transgenic mice. 50-60% of the M3 receptor complexes were plasma membrane associated in human bladder detrusor. Application of PLA for M3 and GPs allowed visualization of M3-GP interactions and revealed individual GP

  13. Dynamics of energy distribution in three channel alpha helix protein based on Davydov’s ansatz

    Energy Technology Data Exchange (ETDEWEB)

    Ahmad, Faozan; Alatas, Husin [Theoretical Physics Division, Department of Physics, Faculty of Mathematics and Sciences Bogor Agricultural University, Bogor, Indonesia, 16680 faozan@ipb.ac.id (Indonesia)

    2015-04-16

    An important aspect of many biological processes at molecular level is the transfer and storage mechanism of bioenergy released in the reaction of the hydrolysis of Adenosinetriphosphate (ATP) by biomacromolecule especially protein. Model of Soliton Davydov is a new break-through that could describe that mechanism. Here we have reformulated quantum mechanical the Davydov theory, using least action principle. Dynamical aspect of the model is analyzed by numerical calculation. We found two dynamical cases: the traveling and pinning soliton that we suggest they are related to the energy transfer and storage mechanism in the protein. Traveling and pinning soliton can be controlled by strength of coupling. In 3- channel approach, we found the breather phenomena in which its frequency is determined by interchannel coupling parameter.

  14. Intracellular trafficking of bio-nanocapsule-liposome complex: Identification of fusogenic activity in the pre-S1 region of hepatitis B virus surface antigen L protein.

    Science.gov (United States)

    Somiya, Masaharu; Sasaki, Yasuo; Matsuzaki, Takashi; Liu, Qiushi; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Maturana, Andrés Daniel; Kuroda, Shun'ichi

    2015-08-28

    Bio-nanocapsules (BNCs) are a hollow nanoparticle consisting of about 100-nm liposome (LP) embedding about 110 molecules of hepatitis B virus (HBV) surface antigen (HBsAg) L protein as a transmembrane protein. Owing to the human hepatocyte-recognizing domains on the N-terminal region (pre-S1 region), BNCs have recently been shown to attach and enter into human hepatic cells using the early infection mechanism of HBV. Since BNCs could form a complex with an LP containing various drugs and genes, BNC-LP complexes have been used as a human hepatic cell-specific drug and gene-delivery system in vitro and in vivo. However, the role of BNCs in cell entry and intracellular trafficking of payloads in BNC-LP complexes has not been fully elucidated. In this study, we demonstrate that low pH-dependent fusogenic activity resides in the N-terminal part of pre-S1 region (NPLGFFPDHQLDPAFG), of which the first FF residues are essential for the activity, and which facilitates membrane fusion between LPs in vitro. Moreover, BNC-LP complexes can bind human hepatic cells specifically, enter into the cells via clathrin-mediated endocytosis, and release their payloads mostly into the cytoplasm. Taken together, the BNC portion of BNC-LP complexes can induce membrane fusion between LPs and endosomal membranes under low pH conditions, and thereby facilitate the endosomal escape of payloads. Furthermore, the fusogenic domain of the pre-S1 region of HBsAg L protein may play a pivotal role in the intracellular trafficking of not only BNC-LP complexes but also of HBV. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. The sea urchin, Paracentrotus lividus, embryo as a "bioethical" model for neurodevelopmental toxicity testing: effects of diazinon on the intracellular distribution of OTX2-like proteins.

    Science.gov (United States)

    Aluigi, M G; Angelini, C; Corte, G; Falugi, C

    2008-12-01

    Presently, a large effort is being made worldwide to increase the sustainability of industrial development, while preserving not only the quality of the environment but also that of animal and human life. In this work, sea urchin early developmental stages were used as a model to test the effects of the organophosphate pesticide (diazinon) on the regulation of gene expression by immunohistochemical localization of the human regulatory protein against the human OTX2. Egg exposure to diazinon did not affect fertilization; however, at concentrations 10(-5)-10(-6) M, it did cause developmental anomalies, among which was the dose-dependent alteration of the intracellular distribution of a regulatory protein that is immunologically related to the human OTX2. The severe anomalies and developmental delay observed after treatment at 10(-5) M concentration are indicators of systemic toxicity, while the results after treatment at 10(-6) M suggest a specific action of the neurotoxic compound. In this second case, exposure to diazinon caused partial delivery of the protein into the nuclei, a defective translocation that particularly affected the blastula and gastrula stages. Therefore, the possibility that neurotoxic agents such as organophosphates may damage embryonic development is taken into account. Specifically, the compounds are known to alter cytoplasmic dynamics, which play a crucial role in regulating the distribution of intracellular structures and molecules, as well as transcription factors. Speculatively, basing our assumptions on Fura2 experiments, we submit the hypothesis that this effect may be due to altered calcium dynamics, which in turn alter cytoskeleton dynamics: the asters, in fact, appear strongly positive to the OTX2 immunoreaction, in both control and exposed samples. Coimmunoprecipitation experiments seem to supply evidence to the hypothesis.

  16. Molecular understanding of sterically controlled compound release through an engineered channel protein (FhuA

    Directory of Open Access Journals (Sweden)

    Hauer Bernhard

    2010-06-01

    Full Text Available Abstract Background Recently we reported a nanocontainer based reduction triggered release system through an engineered transmembrane channel (FhuA Δ1-160; Onaca et al., 2008. Compound fluxes within the FhuA Δ1-160 channel protein are controlled sterically through labeled lysine residues (label: 3-(2-pyridyldithiopropionic-acid-N-hydroxysuccinimide-ester. Quantifying the sterical contribution of each labeled lysine would open up an opportunity for designing compound specific drug release systems. Results In total, 12 FhuA Δ1-160 variants were generated to gain insights on sterically controlled compound fluxes: Subset A six FhuA Δ1-160 variants in which one of the six lysines in the interior of FhuA Δ1-160 was substituted to alanine and Subset B six FhuA Δ1-160 variants in which only one lysine inside the barrel was not changed to alanine. Translocation efficiencies were quantified with the colorimetric TMB (3,3',5,5'-tetramethylbenzidine detection system employing horseradish peroxidase (HRP. Investigation of the six subset A variants identified position K556A as sterically important. The K556A substitution increases TMB diffusion from 15 to 97 [nM]/s and reaches nearly the TMB diffusion value of the unlabeled FhuA Δ1-160 (102 [nM]/s. The prominent role of position K556 is confirmed by the corresponding subset B variant which contains only the K556 lysine in the interior of the barrel. Pyridyl labeling of K556 reduces TMB translocation to 16 [nM]/s reaching nearly background levels in liposomes (13 [nM]/s. A first B-factor analysis based on MD simulations confirmed that position K556 is the least fluctuating lysine among the six in the channel interior of FhuA Δ1-160 and therefore well suited for controlling compound fluxes through steric hindrance. Conclusions A FhuA Δ1-160 based reduction triggered release system has been shown to control the compound flux by the presence of only one inner channel sterical hindrance based on 3

  17. Structural insights into binding of STAC proteins to voltage-gated calcium channels

    Science.gov (United States)

    Wong King Yuen, Siobhan M.; Campiglio, Marta; Tung, Ching-Chieh

    2017-01-01

    Excitation–contraction (EC) coupling in skeletal muscle requires functional and mechanical coupling between L-type voltage-gated calcium channels (CaV1.1) and the ryanodine receptor (RyR1). Recently, STAC3 was identified as an essential protein for EC coupling and is part of a group of three proteins that can bind and modulate L-type voltage-gated calcium channels. Here, we report crystal structures of tandem-SH3 domains of different STAC isoforms up to 1.2-Å resolution. These form a rigid interaction through a conserved interdomain interface. We identify the linker connecting transmembrane repeats II and III in two different CaV isoforms as a binding site for the SH3 domains and report a crystal structure of the complex with the STAC2 isoform. The interaction site includes the location for a disease variant in STAC3 that has been linked to Native American myopathy (NAM). Introducing the mutation does not cause misfolding of the SH3 domains, but abolishes the interaction. Disruption of the interaction via mutations in the II–III loop perturbs skeletal muscle EC coupling, but preserves the ability of STAC3 to slow down inactivation of CaV1.2. PMID:29078335

  18. Channel Protein-Containing Liposomes as Delivery Vehicles for the Controlled Release of Drugs-Optimization of the Lipid Composition

    NARCIS (Netherlands)

    Šmisterová, J.; Deemter, M. van; Schaaf, G. van der; Meijberg, W.; Robillard, G.

    2005-01-01

    In the design of liposomal drug formulations containing a controllable channel protein (MscL), the lipid composition is dictated in part by this membrane protein. This work addresses the question whether therapeutically optimal lipid compositions (phospholipid with high Tm/cholesterol/PEG) are

  19. Channel Catfish, Ictalurus punctatus Rafinesque 1818, Tetraspanin Membrane Protein Family: Characterization and Expression Analysis of CD81 cDNA

    Science.gov (United States)

    CD81, also known as the target of an antiproliferative antibody 1 (TAPA-1), is a member of tetraspanin integral membrane protein family. This protein plays many important roles in immune functions. In this report, we characterized and analyzed expression of the channel catfish CD81 transcript. T...

  20. CPB1 of Aedes aegypti Interacts with DENV2 E Protein and Regulates Intracellular Viral Accumulation and Release from Midgut Cells

    Directory of Open Access Journals (Sweden)

    Hong-Wai Tham

    2014-12-01

    Full Text Available Aedes aegypti is a principal vector responsible for the transmission of dengue viruses (DENV. To date, vector control remains the key option for dengue disease management. To develop new vector control strategies, a more comprehensive understanding of the biological interactions between DENV and Ae. aegypti is required. In this study, a cDNA library derived from the midgut of female adult Ae. aegypti was used in yeast two-hybrid (Y2H screenings against DENV2 envelope (E protein. Among the many interacting proteins identified, carboxypeptidase B1 (CPB1 was selected, and its biological interaction with E protein in Ae. aegypti primary midgut cells was further validated. Our double immunofluorescent assay showed that CPB1-E interaction occurred in the endoplasmic reticulum (ER of the Ae. aegypti primary midgut cells. Overexpression of CPB1 in mosquito cells resulted in intracellular DENV2 genomic RNA or virus particle accumulation, with a lower amount of virus release. Therefore, we postulated that in Ae. aegypti midgut cells, CPB1 binds to the E protein deposited on the ER intraluminal membranes and inhibits DENV2 RNA encapsulation, thus inhibiting budding from the ER, and may interfere with immature virus transportation to the trans-Golgi network.

  1. The Retinoic acid receptor alpha (RARalpha) chimeric proteins PML-, PLZF-, NPM-, and NuMA-RARalpha have distinct intracellular localization patterns.

    Science.gov (United States)

    Hummel, Jeff L; Zhang, Tong; Wells, Richard A; Kamel-Reid, Suzanne

    2002-04-01

    Retinoic acid receptor alpha (RARalpha) gene rearrangement by reciprocal chromosome translocation is the molecular signature of acute promyelocytic leukemia (APL). Disruption of RARalpha function appears to be the likely cause of aberrant myelopoiesis observed in APL, because PML-RARalpha expression has been shown to deregulate the transcription of genes that control myelopoiesis. To target RARalpha chimeric proteins, we engineered epitope-tagged versions of PML-RARalpha, PLZF-RARalpha, NPM-RARalpha, and NuMA-RARalpha (X-RARalphaV5) and generated a panel of stable COS cell lines expressing X-RARalphaV5. Protein fractionation and Western analysis of these COS lines reveal that X-RARalpha proteins localize to both the cytoplasm and nucleus. NPM-RARalpha is predominantly nuclear whereas NuMA-RARalpha is predominantly cytoplasmic. Confocal immunofluorescent microscopy reveals that PML-RARalpha and PLZF-RARalpha share a primarily diffuse nuclear pattern that excludes the nucleolus. NPM-RARalpha is also diffuse in the nucleus but, in contrast to PML-RARalpha and PLZF-RARalpha, is strongly associated with the nucleolus. Strikingly, NuMA-RARalpha predominantly localizes throughout the cytoplasm in a microspeckled pattern. We further demonstrate that NPM and NuMA interact with NPM-RARalpha and NuMA-RARalpha, respectively. The distinct intracellular localization patterns and the shared ability of X-RARalpha to interact with their respective RARalpha partner proteins (X) further support the hypothesis that deregulation of these partners may play a role in APL pathogenesis.

  2. Multiple functions of inositolphosphorylceramides in the formation and intracellular transport of glycosylphosphatidylinositol-anchored proteins in yeast

    OpenAIRE

    Bosson, Régine; Conzelmann, Andreas

    2007-01-01

    The mature sphingolipids of yeast consist of IPCs (inositolphosphorylceramides) and glycosylated derivatives thereof. Beyond being an abundant membrane constituent in the organelles of the secretory pathway, IPCs are also used to constitute the lipid moiety of the majority of GPI (glycosylphosphatidylinositol) proteins, while a minority of GPI proteins contain PI (phosphatidylinositol). Thus all GPI anchor lipids (as well as free IPCs) typically contain C₂₆ fatty acids. However, the primary G...

  3. Clinical spectrum and diagnostic value of antibodies against the potassium channel related protein complex.

    Science.gov (United States)

    Montojo, M T; Petit-Pedrol, M; Graus, F; Dalmau, J

    2015-06-01

    Antibodies against a protein complex that includes voltage-gated potassium channels (VGKC) have been reported in patients with limbic encephalitis, peripheral nerve hyperexcitability, Morvan's syndrome, and a large variety of neurological syndromes. In this article, a review is presented of the syndromes associated with antibodies against VGKC-related proteins and the main antigens of this protein complex, the proteins LGI1 (leucine rich glioma inactivated protein 1) and Caspr2 (contactin-associated protein-like 2). The conceptual problems and clinical implications of the description of antibodies against VGKC-related proteins other than LGI1 and Caspr2 are also discussed. Although initial studies indicated the occurrence of antibodies against VGKC, recent investigations have shown that the main antigens are a neuronal secreted protein known as LGI1 which modulates synaptic excitability, and a protein called Caspr2 located on the cell surface and processes of neurons of different brain regions, and at the juxtaparanodal region of myelinated axons. While antibodies against LGI1 preferentially associate with classical limbic encephalitis, antibodies against Caspr2 associate with a wider spectrum of symptoms, including Morvan's syndrome, peripheral nerve hyperexcitability or neuromyotonia, and limbic or more extensive encephalitis. In addition there are reports of patients with antibodies against VGKC-related proteins that are different from LGI1 or Caspr2. In these cases, the identity and location of the antigens are unknown, the syndrome association is not specific, and the response to treatment uncertain. The discovery of antigens such as LGI1 and Caspr2 has resulted in a clinical and molecular definition of the broad group of diseases previously attributed to antibodies against VGKC. Considering the literature that describes the presence of antibodies against VGKC other than LGI1 and Caspr2 proteins, we propose a practical algorithm for the diagnosis and treatment

  4. PA-6 inhibits inward rectifier currents carried by V93I and D172N gain-of-function KIR2.1 channels, but increases channel protein expression.

    Science.gov (United States)

    Ji, Yuan; Veldhuis, Marlieke G; Zandvoort, Jantien; Romunde, Fee L; Houtman, Marien J C; Duran, Karen; van Haaften, Gijs; Zangerl-Plessl, Eva-Maria; Takanari, Hiroki; Stary-Weinzinger, Anna; van der Heyden, Marcel A G

    2017-07-15

    microscopy demonstrated dose-dependent intracellular K IR 2.1 accumulation following chronic PA-6 application (24 h, 1 and 5 μM). 1) KCNJ2 gain-of-function mutations V93I and D172N in the K IR 2.1 ion channel do not impair PA-6 mediated inhibition of I K1 , 2) PA-6 elevates K IR 2.1 protein expression and induces intracellular K IR 2.1 accumulation, 3) PA-6 is a strong candidate for further preclinical evaluation in treatment of congenital SQT3 and AF.

  5. A Toxoplasma gondii protein with homology to intracellular type Na{sup +}/H{sup +} exchangers is important for osmoregulation and invasion

    Energy Technology Data Exchange (ETDEWEB)

    Francia, Maria E.; Wicher, Sarah [Department of Biological Sciences, University of Idaho, Life Sciences South Room 142, Moscow, ID 83844 (United States); Pace, Douglas A. [Center for Tropical and Emerging Global Diseases and Department of Cellular Biology University of Georgia, Athens, GA 30602 (United States); Sullivan, Jack [Department of Biological Sciences, University of Idaho, Life Sciences South Room 142, Moscow, ID 83844 (United States); Moreno, Silvia N.J. [Center for Tropical and Emerging Global Diseases and Department of Cellular Biology University of Georgia, Athens, GA 30602 (United States); Arrizabalaga, Gustavo, E-mail: gustavo@uidaho.edu [Department of Biological Sciences, University of Idaho, Life Sciences South Room 142, Moscow, ID 83844 (United States)

    2011-06-10

    The obligate intracellular parasite Toxoplasma gondii is exposed to a variety of physiological conditions while propagating in an infected organism. The mechanisms by which Toxoplasma overcomes these dramatic changes in its environment are not known. In yeast and plants, ion detoxification and osmotic regulation are controlled by vacuolar compartments. A novel compartment named the plant-like vacuole or vacuolar compartment (PLV/VAC) has recently been described in T.gondii, which could potentially protect extracellular tachyzoites against salt and other ionic stresses. Here, we report the molecular characterization of the vacuolar type Na{sup +}/H{sup +} exchanger in T. gondii, TgNHE3, and its co-localization with the PLV/VAC proton-pyrophosphatase (TgVP1). We have created a TgNHE3 knockout strain, which is more sensitive to hyperosmotic shock and toxic levels of sodium, possesses a higher intracellular Ca{sup 2+} concentration [Ca{sup 2+}]{sub i}, and exhibits a reduced host invasion efficiency. The defect in invasion correlates with a measurable reduction in the secretion of the adhesin TgMIC2. Overall, our results suggest that the PLV/VAC has functions analogous to those of the vacuolar compartments of plants and yeasts, providing the parasite with a mechanism to resist ionic fluctuations and, potentially, regulate protein trafficking.

  6. Murrayafoline A attenuates the Wnt/{beta}-catenin pathway by promoting the degradation of intracellular {beta}-catenin proteins

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Hyuk; Gwak, Jungsug; Cho, Munju; Ryu, Min-Jung [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of); Lee, Jee-Hyun; Kim, Sang Kyum; Kim, Young Ho [College of Pharmacy, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Lee, Gye Won [Department of Pharmaceutical Engineering, Konyang University, Nonsan 320-711 (Korea, Republic of); Yun, Mi-Young [Department of Beauty Health Care, Daejeon University, Daejeon 305-764 (Korea, Republic of); Cuong, Nguyen Manh [Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, Hanoi (Viet Nam); Shin, Jae-Gook [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of); Song, Gyu-Yong, E-mail: gysong@cnu.ac.kr [College of Pharmacy, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Oh, Sangtaek, E-mail: ohsa@inje.ac.kr [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of)

    2010-01-01

    Molecular lesions in Wnt/{beta}-catenin signaling and subsequent up-regulation of {beta}-catenin response transcription (CRT) occur frequently during the development of colon cancer. To identify small molecules that suppress CRT, we screened natural compounds in a cell-based assay for detection of TOPFalsh reporter activity. Murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa, antagonized CRT that was stimulated by Wnt3a-conditioned medium (Wnt3a-CM) or LiCl, an inhibitor of glycogen synthase kinase-3{beta} (GSK-3{beta}), and promoted the degradation of intracellular {beta}-catenin without altering its N-terminal phosphorylation at the Ser33/37 residues, marking it for proteasomal degradation, or the expression of Siah-1, an E3 ubiquitin ligase. Murrayafoline A repressed the expression of cyclin D1 and c-myc, which is known {beta}-catenin/T cell factor (TCF)-dependent genes and thus inhibited the proliferation of various colon cancer cells. These findings indicate that murrayafoline A may be a potential chemotherapeutic agent for use in the treatment of colon cancer.

  7. Murrayafoline A attenuates the Wnt/β-catenin pathway by promoting the degradation of intracellular β-catenin proteins

    International Nuclear Information System (INIS)

    Choi, Hyuk; Gwak, Jungsug; Cho, Munju; Ryu, Min-Jung; Lee, Jee-Hyun; Kim, Sang Kyum; Kim, Young Ho; Lee, Gye Won; Yun, Mi-Young; Cuong, Nguyen Manh; Shin, Jae-Gook; Song, Gyu-Yong; Oh, Sangtaek

    2010-01-01

    Molecular lesions in Wnt/β-catenin signaling and subsequent up-regulation of β-catenin response transcription (CRT) occur frequently during the development of colon cancer. To identify small molecules that suppress CRT, we screened natural compounds in a cell-based assay for detection of TOPFalsh reporter activity. Murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa, antagonized CRT that was stimulated by Wnt3a-conditioned medium (Wnt3a-CM) or LiCl, an inhibitor of glycogen synthase kinase-3β (GSK-3β), and promoted the degradation of intracellular β-catenin without altering its N-terminal phosphorylation at the Ser33/37 residues, marking it for proteasomal degradation, or the expression of Siah-1, an E3 ubiquitin ligase. Murrayafoline A repressed the expression of cyclin D1 and c-myc, which is known β-catenin/T cell factor (TCF)-dependent genes and thus inhibited the proliferation of various colon cancer cells. These findings indicate that murrayafoline A may be a potential chemotherapeutic agent for use in the treatment of colon cancer.

  8. An electronic channel switching-based aptasensor for ultrasensitive protein detection

    Energy Technology Data Exchange (ETDEWEB)

    Li Hongbo; Wang Cui [State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Wu Zaisheng, E-mail: wuzaisheng@163.com [State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Lu Limin; Qiu Liping; Zhou Hui; Shen Guoli [State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Yu Ruqin, E-mail: rqyu@hnu.edu.cn [State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China)

    2013-01-03

    Highlights: Black-Right-Pointing-Pointer Target IgE is successfully designed to serve as a barrier to separate enzyme from its substrate. Black-Right-Pointing-Pointer This sensing platform of electronic channel switching-based aptasensor can be simply manipulated. Black-Right-Pointing-Pointer The stable hairpin structure of anti-IgE aptamer is utilized to detect target IgE. Black-Right-Pointing-Pointer The sensor is ultrasensitive sensitivity, excellent selectivity and small volume of sample. Black-Right-Pointing-Pointer It is a powerful platform to be further expanded to detect more kinds of proteins and even cells. - Abstract: Due to the ubiquity and essential of the proteins in all living organisms, the identification and quantification of disease-specific proteins are particularly important. Because the conformational change of aptamer upon its target or probe/target/probe sandwich often is the primary prerequisite for the design of an electrochemical aptameric assay system, it is extremely difficult to construct the electrochemical aptasensor for protein assay because the corresponding aptamers cannot often meet the requirement. To circumvent the obstacles mentioned, an electronic channel switching-based (ECS) aptasensor for ultrasensitive protein detection is developed. The essential achievement made is that an innovative sensing concept is proposed: the hairpin structure of aptamer is designed to pull electroactive species toward electrode surface and makes the surface-immobilized IgE serve as a barrier that separates enzyme from its substrate. It seemingly ensures that the ECS aptasensor exhibits most excellent assay features, such as, a detection limit of 4.44 Multiplication-Sign 10{sup -6} {mu}g mL{sup -1} (22.7 fM, 220 zmol in 10-{mu}L sample) (demonstrating a 5 orders of magnitude improvement in detection sensitivity compared with classical electronic aptasensors) and dynamic response range from 4.44 Multiplication-Sign 10{sup -6} to 4.44 Multiplication

  9. Vaccinia virus intracellular enveloped virions move to the cell periphery on microtubules in the absence of the A36R protein.

    Science.gov (United States)

    Herrero-Martínez, Esteban; Roberts, Kim L; Hollinshead, Michael; Smith, Geoffrey L

    2005-11-01

    Vaccinia virus (VACV) intracellular enveloped virus (IEV) particles are transported to the cell periphery on microtubules where they fuse with the plasma membrane to form cell-associated enveloped virus (CEV). Two IEV-specific proteins, F12L and A36R, are implicated in mediating transport of IEV. Without F12L, virus morphogenesis halts after formation of IEV, and CEV is not formed, whereas without A36R, IEV was reported not to be transported, yet CEV was formed, To address the roles of A36R and F12L in IEV transport, viruses with deletions of either F12L (vdeltaF12L) or A36R (vdeltaA36R) were labelled with enhanced green fluorescent protein (EGFP) fused to the core protein A5L, and used to follow CEV production with time. Without F12L, CEV production was inhibited by >99 %, whereas without A36R, CEV were produced at approximately 60 % of wild-type levels at 24 h post-infection. Depolymerization of microtubules, but not actin, inhibited CEV formation in vdeltaA36R-infected cells. Moreover, vdeltaA36R IEV labelled with EGFP fused to the B5R protein co-localized with microtubules, showing that the A36R protein is not required for the interaction of IEV with microtubules. Time-lapse confocal microscopy confirmed that both wild-type and vdeltaA36R IEV moved in a stop-start manner at speeds consistent with microtubular movement, although the mean length of vdeltaA36R IEV movement was shorter. These data demonstrate that VACV IEV is transported to the cell surface using microtubules in the absence of A36R, and therefore IEV must attach to microtubule motors using at least one protein other than A36R.

  10. Effects of protein transduction domain (PTD) selection and position for improved intracellular delivery of PTD-Hsp27 fusion protein formulations.

    Science.gov (United States)

    Ul Ain, Qurrat; Lee, Jong Hwan; Woo, Young Sun; Kim, Yong-Hee

    2016-09-01

    Protein drugs have attracted considerable attention as therapeutic agents due to their diversity and biocompatibility. However, hydrophilic proteins possess difficulty in penetrating lipophilic cell membrane. Although protein transduction domains (PTDs) have shown effectiveness in protein delivery, the importance of selection and position of PTDs in recombinant protein vector constructs has not been investigated. This study intends to investigate the significance of PTD selection and position for therapeutic protein delivery. Heat shock protein 27 (Hsp27) would be a therapeutic protein for the treatment of ischemic heart diseases, but itself is insufficient to prevent systemic degradation and overcoming biochemical barriers during cellular transport. Among all PTD-Hsp27 fusion proteins we cloned, Tat-Hsp27 fusion protein showed the highest efficacy. Nona-arginine (9R) conjugation to the N-terminal of Hsp27 (Hsp27-T) showed higher efficacy than C-terminal. To test the synergistic effect of two PTDs, Tat was inserted to the N-terminal of Hsp27-9R. Tat-Hsp27-9R exhibited enhanced transduction efficiency and significant improvement against oxidative stress and apoptosis. PTD-Hsp27 fusion proteins have strong potential to be developed as therapeutic proteins for the treatment of ischemic heart diseases and selection and position of PTDs for improved efficacy of PTD-fusion proteins need to be optimized considering protein's nature, transduction efficiency and stability.

  11. The Outer Membrane Protein OmpW Forms an Eight-Stranded beta-Barrel with a Hydrophobic Channel

    International Nuclear Information System (INIS)

    Hong, H.; Patel, D.; Tamm, L.; van den Berg, B.

    2006-01-01

    Escherichia coli OmpW belongs to a family of small outer membrane (OM) proteins that are widespread in Gram-negative bacteria. Their functions are unknown, but recent data suggest that they may be involved in the protection of bacteria against various forms of environmental stress. In order to gain insight into the function of these proteins we have determined the crystal structure of Escherichia coli OmpW to 2.7 Angstroms resolution. The structure shows that OmpW forms an eight-stranded beta-barrel with a long and narrow hydrophobic channel that contains a bound LDAO detergent molecule. Single channel conductance experiments show that OmpW functions as an ion channel in planar lipid bilayers. The channel activity can be blocked by the addition of LDAO. Taken together, the data suggest that members of the OmpW family could be involved in the transport of small hydrophobic molecules across the bacterial OM

  12. Effects of amyloid β-peptide fragment 31-35 on the BK channel-mediated K⁺ current and intracellular free Ca²⁺ concentration of hippocampal CA1 neurons.

    Science.gov (United States)

    Zhang, Yu; Shi, Zhi-Gang; Wang, Zhi-Hua; Li, Jian-Guo; Chen, Jin-Yuan; Zhang, Ce

    2014-05-07

    The present study characterizes the effects of Aβ31-35, a short active fragment of amyloid β-peptide (Aβ), upon the BK channel-mediated K⁺ current and intracellular free Ca²⁺ concentration ([Ca²⁺]i) of freshly dissociated pyramidal cells from rat CA1 hippocampus by using whole-cell patch-clamp recording and single cell Ca²⁺ imaging techniques. The results show that: (1) in the presence of voltage- and ATP-gated K⁺ channel blockers application of 5.0 μM Aβ31-35 significantly diminished transient outward K⁺ current amplitudes at clamped voltages between 0 and 45mV; (2) under the same conditions [Ca²⁺]i was minimally affected by 5.0 μM but significantly increased by 12.5 μM and 25 μM Aβ31-35; and (3) when 25 μM of a larger fragment of the amyloid β-peptide, Aβ25-35, was applied, the results were similar to those obtained with the same concentration of Aβ31-35. These results indicate that Aβ31-35 is likely to be the shortest active fragment of the full Aβ sequence, and can be as effectively as the full-length Aβ peptide in suppressing BK-channel mediated K⁺ currents and significantly elevating [Ca²⁺]i in hippocampal CA1 neurons. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  13. Shiga toxin 1 interaction with enterocytes causes apical protein mistargeting through the depletion of intracellular galectin-3

    Energy Technology Data Exchange (ETDEWEB)

    Laiko, Marina; Murtazina, Rakhilya; Malyukova, Irina [Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Zhu, Chengru; Boedeker, Edgar C. [Department of Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131 (United States); Gutsal, Oksana [Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); O' Malley, Robert; Cole, Robert N. [Department of Biochemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Tarr, Phillip I. [Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110 (United States); Murray, Karen F. [Department of Pediatrics, Children' s Hospital and Regional Medical Center, Seattle, WA 98105 (United States); Kane, Anne [The Tufts New England Medical Center, Boston, MA 02111 (United States); Donowitz, Mark [Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Kovbasnjuk, Olga, E-mail: okovbas1@jhmi.edu [Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States)

    2010-02-15

    Shiga toxins (Stx) 1 and 2 are responsible for intestinal and systemic sequelae of infection by enterohemorrhagic Escherichia coli (EHEC). However, the mechanisms through which enterocytes are damaged remain unclear. While secondary damage from ischemia and inflammation are postulated mechanisms for all intestinal effects, little evidence excludes roles for more primary toxin effects on intestinal epithelial cells. We now document direct pathologic effects of Stx on intestinal epithelial cells. We study a well-characterized rabbit model of EHEC infection, intestinal tissue and stool samples from EHEC-infected patients, and T84 intestinal epithelial cells treated with Stx1. Toxin uptake by intestinal epithelial cells in vitro and in vivo causes galectin-3 depletion from enterocytes by increasing the apical galectin-3 secretion. This Shiga toxin-mediated galectin-3 depletion impairs trafficking of several brush border structural proteins and transporters, including villin, dipeptidyl peptidase IV, and the sodium-proton exchanger 2, a major colonic sodium absorptive protein. The mistargeting of proteins responsible for the absorptive function might be a key event in Stx1-induced diarrhea. These observations provide new evidence that human enterocytes are directly damaged by Stx1. Conceivably, depletion of galectin-3 from enterocytes and subsequent apical protein mistargeting might even provide a means whereby other pathogens might alter intestinal epithelial absorption and produce diarrhea.

  14. P2Y receptor-mediated transient relaxation of rat longitudinal ileum preparations involves phospholipase C activation, intracellular Ca(2+) release and SK channel activation.

    Science.gov (United States)

    Mader, Felix; Krause, Ludwig; Tokay, Tursonjan; Hakenberg, Oliver W; Köhling, Rüdiger; Kirschstein, Timo

    2016-05-01

    Purinergic signaling plays a major role in the enteric nervous system, where it governs gut motility through a number of P2X and P2Y receptors. The aim of this study was to investigate the P2Y receptor-mediated motility in rat longitudinal ileum preparations. Ileum smooth muscle strips were prepared from rats, and fixed in an organ bath. Isometric contraction and relaxation responses of the muscle strips were measured with force transducers. Drugs were applied by adding of stock solutions to the organ bath to yield the individual final concentrations. Application of the non-hydrolyzable P2 receptor agonists α,β-Me-ATP or 2-Me-S-ADP (10, 100 μmol/L) dose-dependently elicited a transient relaxation response followed by a sustained contraction. The relaxation response was largely blocked by SK channel blockers apamin (500 nmol/L) and UCL1684 (10 μmol/L), PLC inhibitor U73122 (100 μmol/L), IP3 receptor blocker 2-APB (100 μmol/L) or sarcoendoplasmic Ca(2+) ATPase inhibitor thapsigargin (1 μmol/L), but not affected by atropine, NO synthase blocker L-NAME or tetrodotoxin. Furthermore, α,β-Me-ATP-induced relaxation was suppressed by P2Y1 receptor antagonist MRS2179 (50 μmol/L) or P2Y13 receptor antagonist MRS2211 (100 μmol/L), and was abolished by co-application of the two antagonists, whereas 2-Me-S-ADP-induced relaxation was abolished by P2Y6 receptor antagonist MRS2578 (50 μmol/L). In addition, P2Y1 receptor antagonist MRS2500 (1 μmol/L) not only abolished α,β-Me-ATP-induced relaxation, but also suppressed 2-Me-S-ADP-induced relaxation. P2Y receptor agonist-induced transient relaxation of rat ileum smooth muscle strips is mediated predominantly by P2Y1 receptor, but also by P2Y6 and P2Y13 receptors, and involves PLC, IP3, Ca(2+) release and SK channel activation, but is independent of acetylcholine and NO release.

  15. Negative regulation of opioid receptor-G protein-Ca2+ channel pathway by the nootropic nefiracetam.

    Science.gov (United States)

    Yoshii, Mitsunobu; Furukawa, Taiji; Ogihara, Yoshiyasu; Watabe, Shigeo; Shiotani, Tadashi; Ishikawa, Yasuro; Nishimura, Masao; Nukada, Toshihide

    2004-10-01

    It has recently been reported that nefiracetam, a nootropic agent, is capable of attenuating the development of morphine dependence and tolerance in mice. The mechanism of this antimorphine action is not clear. The present study was designed to address this issue using Xenopus oocytes expressing delta-opioid receptors, G proteins (G(i3alpha) or G(o1alpha)), and N-type (alpha1B) Ca2+ channels. Membrane currents through Ca2+ channels were recorded from the oocytes under voltage-clamp conditions. The Ca2+ channel currents were reduced reversibly by 40-60% in the presence of 1 microM leucine-enkephalin (Leu-Enk). The Leu-Enk-induced current inhibition was recovered promptly by nefiracetam (1 microM), while control currents in the absence of Leu-Enk were not influenced by nefiracetam. A binding assay revealed that 3H-nefiracetam preferentially bound to the membrane fraction of oocytes expressing G(i3alpha). When delta-opioid receptors were coexpressed, the binding was significantly increased. However, an additional expression of alpha1B Ca2+ channels decreased the binding. The results suggest that nefiracetam preferentially binds to G(i3alpha) associated with delta-opioid receptors, thereby inhibiting the association of G proteins with Ca2+ channels. In conclusion, nefiracetam negatively regulates the inhibitory pathway of opioid receptor-G protein-Ca2+ channel.

  16. An Epithelial Ca2+-Sensor Protein is an Alternative to Calmodulin to Compose Functional KCNQ1 Channels

    Directory of Open Access Journals (Sweden)

    Atsushi Inanobe

    2015-07-01

    Full Text Available Background/Aims: KCNQ channels transport K+ ions and participate in various cellular functions. The channels directly assemble with auxiliary proteins such as a ubiquitous Ca2+-sensor protein, calmodulin (CaM, to configure the physiological properties in a tissue-specific manner. Although many CaM-like Ca2+-sensor proteins have been identified in eukaryotes, how KCNQ channels selectively interact with CaM and how the homologues modulate the functionality of the channels remain unclear. Methods: We developed protocols to evaluate the interaction between the green fluorescent protein-tagged C-terminus of KCNQ1 (KCNQ1cL and Ca2+-sensors by detecting its fluorescence in size exclusion chromatography and electrophoresed gels. The effects of Ca2+-sensor proteins on KCNQ1 activity was measured by two electrode voltage clamp technique of Xenopus oocytes. Results: When co-expressed CaM and KCNQ1cL, they assemble in a 4:4 stoichiometry, forming a hetero-octamer. Among nine CaM homologues tested, Calml3 was found to form a hetero-octamer with KCNQ1cL and to associate with the full-length KCNQ1 in a competitive manner with CaM. When co-expressed in oocytes, Calml3 rendered KCNQ1 channels resistant to the voltage-dependent depletion of phosphatidylinositol 4,5-bisphosphate by voltage-sensitive phosphatase. Conclusion: Since Calml3 is closely related to CaM and is prominently expressed in epithelial cells, Calml3 may be a constituent of epithelial KCNQ1 channels and underscores the molecular diversity of endogenous KCNQ1 channels.

  17. An Epithelial Ca2+-Sensor Protein is an Alternative to Calmodulin to Compose Functional KCNQ1 Channels.

    Science.gov (United States)

    Inanobe, Atsushi; Tsuzuki, Chizuru; Kurachi, Yoshihisa

    2015-01-01

    KCNQ channels transport K+ ions and participate in various cellular functions. The channels directly assemble with auxiliary proteins such as a ubiquitous Ca2+- sensor protein, calmodulin (CaM), to configure the physiological properties in a tissue-specific manner. Although many CaM-like Ca2+-sensor proteins have been identified in eukaryotes, how KCNQ channels selectively interact with CaM and how the homologues modulate the functionality of the channels remain unclear. We developed protocols to evaluate the interaction between the green fluorescent protein-tagged C-terminus of KCNQ1 (KCNQ1cL) and Ca2+-sensors by detecting its fluorescence in size exclusion chromatography and electrophoresed gels. The effects of Ca2+-sensor proteins on KCNQ1 activity was measured by two electrode voltage clamp technique of Xenopus oocytes. When co-expressed CaM and KCNQ1cL, they assemble in a 4:4 stoichiometry, forming a hetero-octamer. Among nine CaM homologues tested, Calml3 was found to form a hetero-octamer with KCNQ1cL and to associate with the full-length KCNQ1 in a competitive manner with CaM. When co-expressed in oocytes, Calml3 rendered KCNQ1 channels resistant to the voltage-dependent depletion of phosphatidylinositol 4,5-bisphosphate by voltage-sensitive phosphatase. Since Calml3 is closely related to CaM and is prominently expressed in epithelial cells, Calml3 may be a constituent of epithelial KCNQ1 channels and underscores the molecular diversity of endogenous KCNQ1 channels. Copyright © 2015 S. Karger AG, Basel

  18. Evidence for functional diversity between the voltage-gated proton channel Hv1 and its closest related protein HVRP1.

    Directory of Open Access Journals (Sweden)

    Iris H Kim

    Full Text Available The Hv1 channel and voltage-sensitive phosphatases share with voltage-gated sodium, potassium, and calcium channels the ability to detect changes in membrane potential through voltage-sensing domains (VSDs. However, they lack the pore domain typical of these other channels. NaV, KV, and CaV proteins can be found in neurons and muscles, where they play important roles in electrical excitability. In contrast, VSD-containing proteins lacking a pore domain are found in non-excitable cells and are not involved in neuronal signaling. Here, we report the identification of HVRP1, a protein related to the Hv1 channel (from which the name Hv1 Related Protein 1 is derived, which we find to be expressed primarily in the central nervous system, and particularly in the cerebellum. Within the cerebellar tissue, HVRP1 is specifically expressed in granule neurons, as determined by in situ hybridization and immunohistochemistry. Analysis of subcellular distribution via electron microscopy and immunogold labeling reveals that the protein localizes on the post-synaptic side of contacts between glutamatergic mossy fibers and the granule cells. We also find that, despite the similarities in amino acid sequence and structural organization between Hv1 and HVRP1, the two proteins have distinct functional properties. The high conservation of HVRP1 in vertebrates and its cellular and subcellular localizations suggest an important function in the nervous system.

  19. Circadian rhythm in QT interval is preserved in mice deficient of potassium channel interacting protein 2

    DEFF Research Database (Denmark)

    Gottlieb, Lisa A; Lubberding, Anniek; Larsen, Anders Peter

    2017-01-01

    Potassium Channel Interacting Protein 2 (KChIP2) is suggested to be responsible for the circadian rhythm in repolarization duration, ventricular arrhythmias, and sudden cardiac death. We investigated the hypothesis that there is no circadian rhythm in QT interval in the absence of KChIP2. Implanted...... cardiac deaths were observed. We find similar diurnal (light:dark) and circadian (darkness) rhythms of RR intervals in WT and KChIP2(-/-) mice. Circadian rhythms in QT100 intervals are present in both groups, but at physiological small amplitudes: 1.6 ± 0.2 and 1.0 ± 0.3 ms in WT and KChIP2...

  20. Stapled Voltage-Gated Calcium Channel (CaV) α-Interaction Domain (AID) Peptides Act As Selective Protein-Protein Interaction Inhibitors of CaV Function.

    Science.gov (United States)

    Findeisen, Felix; Campiglio, Marta; Jo, Hyunil; Abderemane-Ali, Fayal; Rumpf, Christine H; Pope, Lianne; Rossen, Nathan D; Flucher, Bernhard E; DeGrado, William F; Minor, Daniel L

    2017-06-21

    For many voltage-gated ion channels (VGICs), creation of a properly functioning ion channel requires the formation of specific protein-protein interactions between the transmembrane pore-forming subunits and cystoplasmic accessory subunits. Despite the importance of such protein-protein interactions in VGIC function and assembly, their potential as sites for VGIC modulator development has been largely overlooked. Here, we develop meta-xylyl (m-xylyl) stapled peptides that target a prototypic VGIC high affinity protein-protein interaction, the interaction between the voltage-gated calcium channel (Ca V ) pore-forming subunit α-interaction domain (AID) and cytoplasmic β-subunit (Ca V β). We show using circular dichroism spectroscopy, X-ray crystallography, and isothermal titration calorimetry that the m-xylyl staples enhance AID helix formation are structurally compatible with native-like AID:Ca V β interactions and reduce the entropic penalty associated with AID binding to Ca V β. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the Ca V α 1 :Ca V β interaction that modulate Ca V function in an Ca V β isoform-selective manner. Together, our studies provide a proof-of-concept demonstration of the use of protein-protein interaction inhibitors to control VGIC function and point to strategies for improved AID-based Ca V modulator design.

  1. Interactions of Oryza sativa OsCONTINUOUS VASCULAR RING-LIKE 1 (OsCOLE1) and OsCOLE1-INTERACTING PROTEIN reveal a novel intracellular auxin transport mechanism.

    Science.gov (United States)

    Liu, Fei; Zhang, Lan; Luo, Yanzhong; Xu, Miaoyun; Fan, Yunliu; Wang, Lei

    2016-10-01

    Little is known about the transport mechanism of intracellular auxin. Here, we report two vacuole-localized proteins, Oryza sativa OsCONTINUOUS VASCULAR RING-LIKE 1 (OsCOLE1) and OsCOLE1-INTERACTING PROTEIN (OsCLIP), that regulate intracellular auxin transport and homoeostasis. Overexpression of OsCOLE1 markedly increased the internode length and auxin content of the stem base, whereas these parameters were decreased in RNA interference (RNAi) plants. OsCOLE1 was localized on the tonoplast and preferentially expressed in mature tissues. We further identified its interacting protein OsCLIP, which was co-localized on the tonoplast. Protein-protein binding assays demonstrated that the N-terminus of OsCOLE1 directly interacted with OsCLIP in yeast cells and the rice protoplast. Furthermore, (3) H-indole-3-acetic acid ((3) H-IAA) transport assays revealed that OsCLIP transported IAA into yeast cells, which was promoted by OsCOLE1. The results indicate that OsCOLE1 affects rice development by regulating intracellular auxin transport through interaction with OsCLIP, which provides a new insight into the regulatory mechanism of intracellular transport of auxin and the roles of vacuoles in plant development. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  2. TmpL, a transmembrane protein required for intracellular redox homeostasis and virulence in a plant and an animal fungal pathogen.

    Directory of Open Access Journals (Sweden)

    Kwang-Hyung Kim

    2009-11-01

    Full Text Available The regulation of intracellular levels of reactive oxygen species (ROS is critical for developmental differentiation and virulence of many pathogenic fungi. In this report we demonstrate that a novel transmembrane protein, TmpL, is necessary for regulation of intracellular ROS levels and tolerance to external ROS, and is required for infection of plants by the necrotroph Alternaria brassicicola and for infection of mammals by the human pathogen Aspergillus fumigatus. In both fungi, tmpL encodes a predicted hybrid membrane protein containing an AMP-binding domain, six putative transmembrane domains, and an experimentally-validated FAD/NAD(P-binding domain. Localization and gene expression analyses in A. brassicicola indicated that TmpL is associated with the Woronin body, a specialized peroxisome, and strongly expressed during conidiation and initial invasive growth in planta. A. brassicicola and A. fumigatus DeltatmpL strains exhibited abnormal conidiogenesis, accelerated aging, enhanced oxidative burst during conidiation, and hypersensitivity to oxidative stress when compared to wild-type or reconstituted strains. Moreover, A. brassicicola DeltatmpL strains, although capable of initial penetration, exhibited dramatically reduced invasive growth on Brassicas and Arabidopsis. Similarly, an A. fumigatus DeltatmpL mutant was dramatically less virulent than the wild-type and reconstituted strains in a murine model of invasive aspergillosis. Constitutive expression of the A. brassicicola yap1 ortholog in an A. brassicicola DeltatmpL strain resulted in high expression levels of genes associated with oxidative stress tolerance. Overexpression of yap1 in the DeltatmpL background complemented the majority of observed developmental phenotypic changes and partially restored virulence on plants. Yap1-GFP fusion strains utilizing the native yap1 promoter exhibited constitutive nuclear localization in the A. brassicicola DeltatmpL background. Collectively, we

  3. Cytoplasmic Domain of MscS Interacts with Cell Division Protein FtsZ: A Possible Non-Channel Function of the Mechanosensitive Channel in Escherichia Coli.

    Directory of Open Access Journals (Sweden)

    Piotr Koprowski

    Full Text Available Bacterial mechano-sensitive (MS channels reside in the inner membrane and are considered to act as emergency valves whose role is to lower cell turgor when bacteria enter hypo-osmotic environments. However, there is emerging evidence that members of the Mechano-sensitive channel Small (MscS family play additional roles in bacterial and plant cell physiology. MscS has a large cytoplasmic C-terminal region that changes its shape upon activation and inactivation of the channel. Our pull-down and co-sedimentation assays show that this domain interacts with FtsZ, a bacterial tubulin-like protein. We identify point mutations in the MscS C-terminal domain that reduce binding to FtsZ and show that bacteria expressing these mutants are compromised in growth on sublethal concentrations of β-lactam antibiotics. Our results suggest that interaction between MscS and FtsZ could occur upon inactivation and/or opening of the channel and could be important for the bacterial cell response against sustained stress upon stationary phase and in the presence of β-lactam antibiotics.

  4. The Aer protein and the serine chemoreceptor Tsr independently sense intracellular energy levels and transduce oxygen, redox, and energy signals for Escherichia coli behavior

    Science.gov (United States)

    Rebbapragada, Anuradha; Johnson, Mark S.; Harding, Gordon P.; Zuccarelli, Anthony J.; Fletcher, Hansel M.; Zhulin, Igor B.; Taylor, Barry L.

    1997-01-01

    We identified a protein, Aer, as a signal transducer that senses intracellular energy levels rather than the external environment and that transduces signals for aerotaxis (taxis to oxygen) and other energy-dependent behavioral responses in Escherichia coli. Domains in Aer are similar to the signaling domain in chemotaxis receptors and the putative oxygen-sensing domain of some transcriptional activators. A putative FAD-binding site in the N-terminal domain of Aer shares a consensus sequence with the NifL, Bat, and Wc-1 signal-transducing proteins that regulate gene expression in response to redox changes, oxygen, and blue light, respectively. A double mutant deficient in aer and tsr, which codes for the serine chemoreceptor, was negative for aerotaxis, redox taxis, and glycerol taxis, each of which requires the proton motive force and/or electron transport system for signaling. We propose that Aer and Tsr sense the proton motive force or cellular redox state and thereby integrate diverse signals that guide E. coli to environments where maximal energy is available for growth. PMID:9380671

  5. Patch clamp and perfusion techniques for studying ion channels expressed in Xenopus oocytes.

    Science.gov (United States)

    Yang, Junqiu; Delaloye, Kelli; Lee, Urvi S; Cui, Jianmin

    2011-01-10

    The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca(2+)-activated K(+) (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release. BK channels are activated by membrane depolarization and by intracellular Ca(2+) and Mg(2+). Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18°C. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

  6. Serotonin storage pools in basophil leukemia and mast cells: characterization of two types of serotonin binding protein and radioautographic analysis of the intracellular distribution of [3H]serotonin

    International Nuclear Information System (INIS)

    Tamir, H.; Theoharides, T.C.; Gershon, M.D.; Askenase, P.W.

    1982-01-01

    The binding of serotonin to protein(s) derived from rat basophil leukemia (RBL) cells and mast cells was studied. Two types of serotonin binding protein in RBL cells was found. These proteins differed from one another in molecular weight and eluted in separate peaks from sephadex G-200 columns. Peak I protein (KD = 1.9 x 10 -6 M) was a glycoprotein that bound to concanavalin A (Con A); Peak II protein (KD 1 = 4.5 x 10 - 8 M; KD 2 = 3.9 x 10 -6 M) did not bind to Con A. Moreover, binding of [ 3 H]serotonin to protein of Peak I was sensitive to inhibition by reserpine, while binding of [ 3 H]serotonin to protein of Peak II resisted inhibition by that drug. Other differences between the two types of binding protein were found, the most significant of which was the far more vigorous conditions of homogenization required to extract Peak I than Peak II protein. Electron microscope radioautographic analysis of the intracellular distribution of [ 3 H] serotonin taken up in vitro by RBL cells or in vivo by murine mast cells indicated that essentially all of the labeled amine was located in cytoplasmic granules.No evidence for a pool in the cytosol was found and all granules were capable of becoming labeled. The presence of two types of intracellular serotonin binding proteins in these cells may indicate that there are two intracellular storage compartments for the amine. Both may be intragranular, but Peak I protein may be associated with the granular membrane while Peak II protein may be more free within the granular core. Different storage proteins may help to explain the differential release of amines from mast cell granules

  7. Functional genomics of intracellular bacteria.

    Science.gov (United States)

    de Barsy, Marie; Greub, Gilbert

    2013-07-01

    During the genomic era, a large amount of whole-genome sequences accumulated, which identified many hypothetical proteins of unknown function. Rapidly, functional genomics, which is the research domain that assign a function to a given gene product, has thus been developed. Functional genomics of intracellular pathogenic bacteria exhibit specific peculiarities due to the fastidious growth of most of these intracellular micro-organisms, due to the close interaction with the host cell, due to the risk of contamination of experiments with host cell proteins and, for some strict intracellular bacteria such as Chlamydia, due to the absence of simple genetic system to manipulate the bacterial genome. To identify virulence factors of intracellular pathogenic bacteria, functional genomics often rely on bioinformatic analyses compared with model organisms such as Escherichia coli and Bacillus subtilis. The use of heterologous expression is another common approach. Given the intracellular lifestyle and the many effectors that are used by the intracellular bacteria to corrupt host cell functions, functional genomics is also often targeting the identification of new effectors such as those of the T4SS of Brucella and Legionella.

  8. Intracellular mediators of potassium-induced aldosterone secretion

    International Nuclear Information System (INIS)

    Ganguly, A.; Chiou, S.; Davis, J.S.

    1990-01-01

    We have investigated the intracellular messengers of potassium in eliciting aldosterone secretion in calf adrenal glomerulosa cells since there were unresolved issues relating to the role of phosphoinositides, cAMP and protein kinases. We observed no evidence of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) in 3 H-inositol labeled alf adrenal cells or increase of cAMP in response to potassium. Addition of calcium channel blocker, nitrendipine after stimulating adrenal glomerulosa cells with potassium, markedly inhibited aldosterone secretion. A calmodulin inhibitor (W-7) produced greater reduction of aldosterone secretion than an inhibitor of protein kinase C (H-7). These results suggest that a rise in cytosolic free calcium concentration through voltage-dependent calcium channel and calmodulin are the critical determinants of aldosterone secretion stimulated by potassium

  9. Handheld mechanical cell lysis chip with ultra-sharp silicon nano-blade arrays for rapid intracellular protein extraction.

    Science.gov (United States)

    Yun, Sung-Sik; Yoon, Sang Youl; Song, Min-Kyung; Im, Sin-Hyeog; Kim, Sohee; Lee, Jong-Hyun; Yang, Sung

    2010-06-07

    This paper presents a handheld mechanical cell lysis chip with ultra-sharp nano-blade arrays fabricated by simple and cost effective crystalline wet etching of (110) silicon. The ultra-sharp nano-blade array is simply formed by the undercutting of (110) silicon during the crystalline wet etching process. Cells can be easily disrupted by the silicon nano-blade array without the help of additional reagents or electrical sources. Based on the bench-top test of the proposed device, a handheld mechanical cell lysis chip with the nano-blade arrays is designed and fabricated for direct connection to a commercial syringe. The direct connection to a syringe provides rapid cell lysis, easy handling, and minimization of the lysate dead volume. The protein concentration in the cell lysate obtained by the proposed lysis chip is quantitatively comparable to the one prepared by a conventional chemical lysis method.

  10. Inner tegument proteins of Herpes Simplex Virus are sufficient for intracellular capsid motility in neurons but not for axonal targeting

    Science.gov (United States)

    Müller, Oliver; Ivanova, Lyudmila; Bialy, Dagmara; Pohlmann, Anja; Binz, Anne; Hegemann, Maike; Viejo-Borbolla, Abel; Rosenhahn, Bodo; Bauerfeind, Rudolf; Sodeik, Beate

    2017-01-01

    Upon reactivation from latency and during lytic infections in neurons, alphaherpesviruses assemble cytosolic capsids, capsids associated with enveloping membranes, and transport vesicles harboring fully enveloped capsids. It is debated whether capsid envelopment of herpes simplex virus (HSV) is completed in the soma prior to axonal targeting or later, and whether the mechanisms are the same in neurons derived from embryos or from adult hosts. We used HSV mutants impaired in capsid envelopment to test whether the inner tegument proteins pUL36 or pUL37 necessary for microtubule-mediated capsid transport were sufficient for axonal capsid targeting in neurons derived from the dorsal root ganglia of adult mice. Such neurons were infected with HSV1-ΔUL20 whose capsids recruited pUL36 and pUL37, with HSV1-ΔUL37 whose capsids associate only with pUL36, or with HSV1-ΔUL36 that assembles capsids lacking both proteins. While capsids of HSV1-ΔUL20 were actively transported along microtubules in epithelial cells and in the somata of neurons, those of HSV1-ΔUL36 and -ΔUL37 could only diffuse in the cytoplasm. Employing a novel image analysis algorithm to quantify capsid targeting to axons, we show that only a few capsids of HSV1-ΔUL20 entered axons, while vesicles transporting gD utilized axonal transport efficiently and independently of pUL36, pUL37, or pUL20. Our data indicate that capsid motility in the somata of neurons mediated by pUL36 and pUL37 does not suffice for targeting capsids to axons, and suggest that capsid envelopment needs to be completed in the soma prior to targeting of herpes simplex virus to the axons, and to spreading from neurons to neighboring cells. PMID:29284065

  11. Electrochemical impedance spectroscopy for black lipid membranes fused with channel protein supported on solid-state nanopore.

    Science.gov (United States)

    Khan, Muhammad S; Dosoky, Noura S; Berdiev, Bakhrom K; Williams, John D

    2016-12-01

    Black lipid membranes (BLMs) have been used for detecting single-channel activities of pore-forming peptides and ion channels. However, the short lifetimes and poor mechanical stability of suspended bilayers limit their applications in high throughput electrophysiological experiments. In this work, we present a synthetic solid-state nanopore functionalized with BLM fused with channel protein. A nanopore with diameter of ~180 nm was electrochemically fabricated in a thin silicon membrane. Folding and painting techniques were demonstrated for production of stable suspended BLMs followed by incorporation of transmembrane protein, ENaC. Membrane formation was confirmed by employing electrochemical impedance spectroscopy (EIS) in the frequency regime of 10 -2 -10 5  Hz. Results show that electrochemically fabricated solid state nanopore support resulted in excellent membrane stability, with >1 GΩ of up to 72 and 41 h for painting and folding techniques, respectively. After fusion of ENaC channel protein, the BLM exhibits the stability of ~5 h. We anticipate that such a solid-state nanopore with diameter in the range of 150-200 nm and thickness <1 µm could be a potential platform to enhance the throughput of ion-channel characterization using BLMs.

  12. RIM proteins tether Ca2+-channels to presynaptic active zones via a direct PDZ-domain interaction

    Science.gov (United States)

    Kaeser, Pascal S.; Deng, Lunbin; Wang, Yun; Dulubova, Irina; Liu, Xinran; Rizo, Josep; Südhof, Thomas C.

    2011-01-01

    SUMMARY At a synapse, fast synchronous neurotransmitter release requires localization of Ca2+-channels to presynaptic active zones. How Ca2+-channels are recruited to active zones, however, remains unknown. Using unbiased yeast two-hybrid screens, we here identify a direct interaction of the central PDZ-domain of the active-zone protein RIM with the C-termini of presynaptic N- and P/Q-type Ca2+-channels, but not L-type Ca2+-channels. To test the physiological significance of this interaction, we generated conditional knockout mice lacking all presynaptic RIM isoforms. Deletion of all RIMs ablated most neurotransmitter release by simultaneously impairing the priming of synaptic vesicles and by decreasing the presynaptic localization of Ca2+-channels. Strikingly, rescue of the decreased Ca2+-channel localization required the RIM PDZ-domain, whereas rescue of vesicle priming required the RIM N-terminus. We propose that RIMs tether N- and P/Q-type Ca2+-channels to presynaptic active zones via a direct PDZ-domain mediated interaction, thereby enabling fast, synchronous triggering of neurotransmitter release at a synapse. PMID:21241895

  13. Effects of fluoxetine on protein expression of potassium ion channels in the brain of chronic mild stress rats

    Directory of Open Access Journals (Sweden)

    Chunlin Chen

    2015-01-01

    Full Text Available The purpose of this study is to investigate the expression of major potassium channel subtypes in the brain of chronical mild stress (CMS rats and reveal the effects of fluoxetine on the expression of these channels. Rats were exposed to a variety of unpredictable stress for three weeks and induced anhedonia, lower sucrose preference, locomotor activity and lower body weight. The protein expressions were determined by Western blot. CMS significantly increased the expression of Kv2.1 channel in frontal cortex but not in hippocampus, and the expression level was normalized after fluoxetine treatment. The expression of TREK-1 channel was also obviously increased in frontal cortex in CMS rats. Fluoxetine treatment might prevent this increase. However, the expression of Kv3.1 and Kv4.2 channels was considerably decreased in hippocampus after CMS, and was not affected by fluoxetine. These results suggest that different subtypes of potassium channels are associated with the pathophysiology of depression and that the therapeutical effects of fluoxetine may relate to Kv2.1 and TREK-1 potassium channels.

  14. Fibroblast growth factor type 1 receptor stimulation of T-type Ca2+channels in sensory neurons requires the phosphatidylinositol 3-kinase and protein kinase A pathways, independently of Akt.

    Science.gov (United States)

    Si, Weibing; Zhang, Yuan; Chen, Kun; Hu, Dan; Qian, Zhiyuan; Gong, Shan; Li, Hua; Hao, Yuefeng; Tao, Jin

    2018-01-31

    Fibroblast growth factor-23 (FGF-23) secreted by osteocytes is known as a circulating factor that is essential for phosphate homeostasis. Recent studies have implicated FGF-23 in the nociceptive signalling of peripheral sensory neurons. However, the relevant mechanisms underlying this effect are not known. In this study, we determine the role of FGF-23 in regulating T-type Ca 2+ channels (T-type channels) in small-diameter dorsal root ganglion (DRG) neurons in mice. Our results show that FGF-23 increases T-type channel currents in a concentration-dependent manner. This FGF-23-induced response was dependent on FGF type 1 receptor (FGFR1) and was accompanied by a depolarizing shift in the steady-state inactivation curve. Pretreatment of neurons with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 prevented the FGF-23-mediated T-type channel response. Analysis of phospho-Akt (p-Akt) revealed that FGF-23 significantly activated Akt, but Akt inhibition did not affect the FGF-23-induced T-type channel current increase. The cell-permeable protein kinase A (PKA) inhibitor KT-5720 pretreatment and intracellular application of PKI 6-22 both abolished the stimulatory effects of FGF-23 on T-type channels, but inhibition of PKC had no effect. In summary, these findings indicate that FGF-23 stimulates T-type channel activity via activation of FGFR1, which is coupled to the PI3K-dependent PKA signalling cascade in small DRG neurons. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Expression and high glucose-mediated regulation of K+ channel interacting protein 3 (KChIP3) and KV4 channels in retinal Müller glial cells.

    Science.gov (United States)

    Chavira-Suárez, Erika; Sandoval, Alejandro; Felix, Ricardo; Lamas, Mónica

    2011-01-14

    Normal vision depends on the correct function of retinal neurons and glia and it is impaired in the course of diabetic retinopathy. Müller cells, the main glial cells of the retina, suffer morphological and functional alterations during diabetes participating in the pathological retinal dysfunction. Recently, we showed that Müller cells express the pleiotropic protein potassium channel interacting protein 3 (KChIP3), an integral component of the voltage-gated K(+) channels K(V)4. Here, we sought to analyze the role of KChIP3 in the molecular mechanisms underlying hyperglycemia-induced phenotypic changes in the glial elements of the retina. The expression and function of KChIp3 was analyzed in vitro in rat Müller primary cultures grown under control (5.6 mM) or high glucose (25 mM) (diabetic-like) conditions. We show the up-regulation of KChIP3 expression in Müller cell cultures under high glucose conditions and demonstrate a previously unknown interaction between the K(V)4 channel and KChIP3 in Müller cells. We show evidence for the expression of a 4-AP-sensitive transient outward voltage-gated K(+) current and an alteration in the inactivation of the macroscopic outward K(+) currents expressed in high glucose-cultured Müller cells. Our data support the notion that induction of KChIP3 and functional changes of K(V)4 channels in Müller cells could exert a physiological role in the onset of diabetic retinopathy. Copyright © 2010 Elsevier Inc. All rights reserved.

  16. Intracellular Localization of the Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein: Absence of Nucleolar Accumulation during Infection and after Expression as a Recombinant Protein in Vero Cells

    OpenAIRE

    Rowland, Raymond R. R.; Chauhan, Vinita; Fang, Ying; Pekosz, Andrew; Kerrigan, Maureen; Burton, Miriam D.

    2005-01-01

    The nucleocapsid (N) protein of several members within the order Nidovirales localizes to the nucleolus during infection and after transfection of cells with N genes. However, confocal microscopy of N protein localization in Vero cells infected with the severe acute respiratory syndrome coronavirus (SARS-CoV) or transfected with the SARS-CoV N gene failed to show the presence of N in the nucleoplasm or nucleolus. Amino acids 369 to 389, which contain putative nuclear localization signal (NLS)...

  17. Protein self-assembly and lipid binding in the folding of the potassium channel KcsA

    NARCIS (Netherlands)

    Barrera, F.N.; Renard, M.L.; Poveda, J.A.; de Kruijff, B.; Killian, J.A.; González-Ros, J.M.

    2008-01-01

    Moderate concentrations of the alcohol 2,2,2-trifluoroethanol (TFE) cause the coupled unfolding and dissociation into subunits of the homotetrameric potassium channel KcsA, in a process that is partially irreversible when the protein is solubilized in plain dodecyl â-D-maltoside (DDM) micelles

  18. Live-cell visualization of intracellular interaction between a nuclear migration protein (hNUDC and the thrombopoietin receptor (Mpl.

    Directory of Open Access Journals (Sweden)

    Yuan-Bin Zheng

    Full Text Available We previously demonstrated that endogenous hNUDC and Mpl co-localized in the perinuclear and cytoplasmic regions of megakaryocyte cells by indirect immunofluorescence. We further reported that hNUDC accumulated in the Golgi when NIH 3T3 cells were transfected with an hNUDC expression vector alone. However, co-transfection with hNUDC and Mpl expression vectors caused both proteins to co-localize predominantly in the cytosol. These observations led us to hypothesize that a complex containing hNUDC and Mpl may alter hNUDC subcellular location and induce its secretion. In the present study, we test this hypothesis by employing bimolecular fluorescence complementation (BiFC to detect and visualize the complex formation of hNUDC/Mpl in living cells. We further examined in detail the subcellular locations of the hNUDC/Mpl complex by co-transfection of BiFC chimeras with known subcellular markers. The distribution of hNUDC/Mpl in the endoplasmic reticulum (ER, Golgi and cell surface was determined. Furthermore, the N-terminal 159 amino acids of hNUDC, but not C-terminal half, bound to Mpl in vivo and exhibited a similar localization pattern to that of full-length hNUDC in Cos-1 cells. Adenovirus-mediated overexpression of hNUDC or its N-terminal 159 residues in a human megakaryocyte cell line (Dami resulted in increased levels of hNUDC or hNUDC(1-159 secretion. In contrast, depletion of Mpl by transfecting Dami cells with adenovirus bearing Mpl-targeting siRNA significantly blocked hNUDC secretion. Thus, we provide the first evidence that the N-terminal region of hNUDC contains all of the necessary information to complex with Mpl and traffic through the secretory pathway.

  19. Interaction of poxvirus intracellular mature virion proteins with the TPR domain of kinesin light chain in live infected cells revealed by two-photon-induced fluorescence resonance energy transfer fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    Jeshtadi, Ananya; Burgos, Pierre; Stubbs, Christopher D; Parker, Anthony W; King, Linda A; Skinner, Michael A; Botchway, Stanley W

    2010-12-01

    Using two-photon-induced fluorescence lifetime imaging microscopy, we corroborate an interaction (previously demonstrated by yeast two-hybrid domain analysis) of full-length vaccinia virus (VACV; an orthopoxvirus) A36 protein with the cellular microtubule motor protein kinesin. Quenching of enhanced green fluorescent protein (EGFP), fused to the C terminus of VACV A36, by monomeric red fluorescent protein (mDsRed), fused to the tetratricopeptide repeat (TPR) domain of kinesin, was observed in live chicken embryo fibroblasts infected with either modified vaccinia virus Ankara (MVA) or wild-type fowlpox virus (FWPV; an avipoxvirus), and the excited-state fluorescence lifetime of EGFP was reduced from 2.5 ± 0.1 ns to 2.1 ± 0.1 ns due to resonance energy transfer to mDsRed. FWPV does not encode an equivalent of intracellular enveloped virion surface protein A36, yet it is likely that this virus too must interact with kinesin to facilitate intracellular virion transport. To investigate possible interactions between innate FWPV proteins and kinesin, recombinant FWPVs expressing EGFP fused to the N termini of FWPV structural proteins Fpv140, Fpv168, Fpv191, and Fpv198 (equivalent to VACV H3, A4, p4c, and A34, respectively) were generated. EGFP fusions of intracellular mature virion (IMV) surface protein Fpv140 and type II membrane protein Fpv198 were quenched by mDsRed-TPR in recombinant FWPV-infected cells, indicating that these virion proteins are found within 10 nm of mDsRed-TPR. In contrast, and as expected, EGFP fusions of the IMV core protein Fpv168 did not show any quenching. Interestingly, the p4c-like protein Fpv191, which demonstrates late association with preassembled IMV, also did not show any quenching.

  20. Expression of tetraspan protein CD63 activates protein-tyrosine kinase (PTK) and enhances the PTK-induced inhibition of ROMK channels.

    NARCIS (Netherlands)

    Lin, D.; Kamsteeg, E.J.; Zhang, Y.; Jin, Y.; Sterling, H.; Yue, P.; Roos, M.; Duffield, A.; Spencer, J.; Caplan, M.; Wang, W.H.

    2008-01-01

    In the present study, we tested the role of CD63 in regulating ROMK1 channels by protein-tyrosine kinase (PTK). Immunocytochemical staining shows that CD63 and receptor-linked tyrosine phosphatase alpha (RPTPalpha) are expressed in the cortical collecting duct and outer medulla collecting duct.

  1. Characterization and intracellular localization of the Epstein-Barr virus protein BFLF2: interactions with BFRF1 and with the nuclear lamina.

    Science.gov (United States)

    Gonnella, Roberta; Farina, Antonella; Santarelli, Roberta; Raffa, Salvatore; Feederle, Regina; Bei, Roberto; Granato, Marisa; Modesti, Andrea; Frati, Luigi; Delecluse, Henri-Jacques; Torrisi, Maria Rosaria; Angeloni, Antonio; Faggioni, Alberto

    2005-03-01

    We have reported in the accompanying paper that the BFRF1 protein of Epstein-Barr virus (EBV) is important for efficient primary viral envelopment and egress (A. Farina, R. Feederle, S. Raffa, R. Gonnella, R. Santarelli, L. Frati, A. Angeloni, M. R. Torrisi, A. Faggioni, and H.-J. Delecluse, J. Virol. 79:3703-3712). Here we describe the characterization of the product of the EBV BFLF2 gene, which belongs to a family of conserved herpesviral genes which include the UL31 genes of herpes simplex virus and of pseudorabies virus and whose products are known to interact with UL34, the positional homolog of BFRF1. BFLF2 is an early transcript and is expressed in a variety of cell lines upon EBV lytic cycle activation. Western blotting of purified virion preparations showed that BFLF2 is a component of intracellular virions but is absent from mature extracellular virions. Coimmunoprecipitation experiments indicated that BFLF2 interacts with BFRF1, which was confirmed by immunofluorescence confocal microscopy showing that the two proteins colocalize on the nuclear membrane not only upon cotransfection in epithelial cells but also during viral replication. In cells carrying an EBV mutant with the BFRF1 gene deleted (293-BFRF1-KO cells) BFLF2 expression was low, and it was restored to wild-type levels upon treatment of the cells with the proteasome inhibitor MG132. Furthermore, recomplementing the 293-BFRF1-KO cells by BFRF1 transfection restored BFLF2 expression to the wild-type level. In addition, when expressed alone BFLF2 was localized diffusely inside the nucleus, whereas in the presence of BFRF1 the two proteins colocalized at the nuclear rim. Finally, 293 epithelial cells transfected with either protein or cotransfected were analyzed by electron microscopy to investigate potential alterations in the morphology of the nuclear membrane. The ultrastructural analysis revealed that (i) BFRF1 caused duplications of the nuclear membrane, similar to those reported to occur

  2. Initial evaluation of protein A modified capillary-channeled polymer fibers for the capture and recovery of immunoglobulin G.

    Science.gov (United States)

    Schadock-Hewitt, Abby J; Marcus, R Kenneth

    2014-03-01

    A novel protein A affinity chromatography stationary phase has been developed from polypropylene capillary-channeled polymer fibers modified with a recombinant protein A ligand for the capture and recovery of immunoglobulin G (IgG) with high specificity and yield. An SPE micropipette tip format was employed so that solvent, protein, and antibody consumption was minimized. The adsorption modification of the fiber surfaces with protein A was evaluated as a function of feed concentration and volume. Optimal modification of the fiber surface with protein A yielded a 5.7 mg/mL (bed volume) ligand capacity with the modified fibers showing stability across numerous solvent environments. Performance was evaluated through exposure to human IgG and myoglobin, individually and as a mixture. Myoglobin was used as a surrogate for host cell proteins common to growth media. The efficacy of the selective binding to the ligand is demonstrated by the 2.9:1 (IgG/protein A) binding stoichiometry. Elution with 0.1 M acetic acid yielded an 89% recovery of the captured IgG based on absorption measurements of the collected eluents. Regeneration was possible with 10 mM NaOH. Protein A modified polypropylene capillary-channeled polymer fibers show promising initial results as an affinity phase for efficient capture and purification of IgG. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Tetrameric far-red fluorescent protein as a scaffold to assemble an octavalent peptide nanoprobe for enhanced tumor targeting and intracellular uptake in vivo.

    Science.gov (United States)

    Luo, Haiming; Yang, Jie; Jin, Honglin; Huang, Chuan; Fu, Jianwei; Yang, Fei; Gong, Hui; Zeng, Shaoqun; Luo, Qingming; Zhang, Zhihong

    2011-06-01

    Relatively weak tumor affinities and short retention time in vivo hinder the application of targeting peptides in tumor molecular imaging. Multivalent strategies based on various scaffolds have been utilized to improve the ability of peptide-receptor binding or extend the clearance time of peptide-based probes. Here, we use a tetrameric far-red fluorescent protein (tfRFP) as a scaffold to create a self-assembled octavalent peptide fluorescent nanoprobe (Octa-FNP) using a genetic engineering approach. The multiligand connecting, fluorophore labeling and nanostructure formation of Octa-FNP were performed in one step. In vitro studies showed Octa-FNP is a 10-nm fluorescent probe with excellent serum stability. Cellular uptake of Octa-FNP by human nasopharyngeal cancer 5-8F cells is 15-fold of tetravalent probe, ∼80-fold of monovalent probe and ∼600-fold of nulvalent tfRFP. In vivo enhanced tumor targeting and intracellular uptake of Octa-FNP were confirmed using optical imaging and Western blot analysis. It achieved extremely high contrast of Octa-FNP signal between tumor tissue and normal organs, especially seldom Octa-FNP detected in liver and spleen. Owing to easy preparation, precise structural and functional control, and multivalent effect, Octa-FNP provides a powerful tool for tumor optical molecular imaging and evaluating the targeting ability of numerous peptides in vivo.

  4. Development of supported biomimetic membranes for insertion of aquaporin protein water channels for novel water filtration applications

    DEFF Research Database (Denmark)

    Hansen, Jesper Søndergaard

    Aquaporins represent a class of membrane protein channels found in all living organisms that selectively transport water molecules across biological membranes. The work presented in this thesis was motivated by the conceptual idea of incorporating aquaporin water channels into biomimetic membranes...... to develop novel water separation technologies. To accomplish this, it is necessary to construct an efficient platform to handle biomimetic membranes. Moreover, general methods are required to reliable and controllable reconstitute membrane proteins into artificially made model membranes....... These are the topics of this thesis, and are divided into three main chapters. Chapter 2 reviews recent advances in the design and construction of biomimetic membrane arrays. Moreover, current and novel strategies for the reconstitution of membrane proteins into biomimetic membranes are reviewed. Chapter 3 presents...

  5. Xanthurenic acid binds to neuronal G-protein-coupled receptors that secondarily activate cationic channels in the cell line NCB-20.

    Directory of Open Access Journals (Sweden)

    Omar Taleb

    Full Text Available Xanthurenic acid (XA is a metabolite of the tryptophan oxidation pathway through kynurenine and 3-hydroxykynurenine. XA was until now considered as a detoxification compound and dead-end product reducing accumulation of reactive radical species. Apart from a specific role for XA in the signaling cascade resulting in gamete maturation in mosquitoes, nothing was known about its functions in other species including mammals. Based upon XA distribution, transport, accumulation and release in the rat brain, we have recently suggested that XA may potentially be involved in neurotransmission/neuromodulation, assuming that neurons presumably express specific XA receptors. Recently, it has been shown that XA could act as a positive allosteric ligand for class II metabotropic glutamate receptors. This finding reinforces the proposed signaling role of XA in brain. Our present results provide several lines of evidence in favor of the existence of specific receptors for XA in the brain. First, binding experiments combined with autoradiography and time-course analysis led to the characterization of XA binding sites in the rat brain. Second, specific kinetic and pharmacological properties exhibited by these binding sites are in favor of G-protein-coupled receptors (GPCR. Finally, in patch-clamp and calcium imaging experiments using NCB-20 cells that do not express glutamate-induced calcium signals, XA elicited specific responses involving activation of cationic channels and increases in intracellular Ca(2+ concentration. Altogether, these results suggest that XA, acting through a GPCR-induced cationic channel modulatory mechanism, may exert excitatory functions in various brain neuronal pathways.

  6. Role of phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) in intracellular amyloid precursor protein (APP) processing and amyloid plaque pathogenesis.

    Science.gov (United States)

    Xiao, Qingli; Gil, So-Chon; Yan, Ping; Wang, Yan; Han, Sharon; Gonzales, Ernie; Perez, Ronaldo; Cirrito, John R; Lee, Jin-Moo

    2012-06-15

    One of the pathological hallmarks of Alzheimer disease is the accumulation of amyloid plaques in the extracellular space in the brain. Amyloid plaques are primarily composed of aggregated amyloid β peptide (Aβ), a proteolytic fragment of the transmembrane amyloid precursor protein (APP). For APP to be proteolytically cleaved into Aβ, it must be internalized into the cell and trafficked to endosomes where specific protease complexes can cleave APP. Several recent genome-wide association studies have reported that several single nucleotide polymorphisms (SNPs) in the phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) gene were significantly associated with Alzheimer disease, suggesting a role in APP endocytosis and Aβ generation. Here, we show that PICALM co-localizes with APP in intracellular vesicles of N2a-APP cells after endocytosis is initiated. PICALM knockdown resulted in reduced APP internalization and Aβ generation. Conversely, PICALM overexpression increased APP internalization and Aβ production. In vivo, PICALM was found to be expressed in neurons and co-localized with APP throughout the cortex and hippocampus in APP/PS1 mice. PICALM expression was altered using AAV8 gene transfer of PICALM shRNA or PICALM cDNA into the hippocampus of 6-month-old APP/PS1 mice. PICALM knockdown decreased soluble and insoluble Aβ levels and amyloid plaque load in the hippocampus. Conversely, PICALM overexpression increased Aβ levels and amyloid plaque load. These data indicate that PICALM, an adaptor protein involved in clathrin-mediated endocytosis, regulates APP internalization and subsequent Aβ generation. PICALM contributes to amyloid plaque load in brain likely via its effect on Aβ metabolism.

  7. Role of Phosphatidylinositol Clathrin Assembly Lymphoid-Myeloid Leukemia (PICALM) in Intracellular Amyloid Precursor Protein (APP) Processing and Amyloid Plaque Pathogenesis*

    Science.gov (United States)

    Xiao, Qingli; Gil, So-Chon; Yan, Ping; Wang, Yan; Han, Sharon; Gonzales, Ernie; Perez, Ronaldo; Cirrito, John R.; Lee, Jin-Moo

    2012-01-01

    One of the pathological hallmarks of Alzheimer disease is the accumulation of amyloid plaques in the extracellular space in the brain. Amyloid plaques are primarily composed of aggregated amyloid β peptide (Aβ), a proteolytic fragment of the transmembrane amyloid precursor protein (APP). For APP to be proteolytically cleaved into Aβ, it must be internalized into the cell and trafficked to endosomes where specific protease complexes can cleave APP. Several recent genome-wide association studies have reported that several single nucleotide polymorphisms (SNPs) in the phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) gene were significantly associated with Alzheimer disease, suggesting a role in APP endocytosis and Aβ generation. Here, we show that PICALM co-localizes with APP in intracellular vesicles of N2a-APP cells after endocytosis is initiated. PICALM knockdown resulted in reduced APP internalization and Aβ generation. Conversely, PICALM overexpression increased APP internalization and Aβ production. In vivo, PICALM was found to be expressed in neurons and co-localized with APP throughout the cortex and hippocampus in APP/PS1 mice. PICALM expression was altered using AAV8 gene transfer of PICALM shRNA or PICALM cDNA into the hippocampus of 6-month-old APP/PS1 mice. PICALM knockdown decreased soluble and insoluble Aβ levels and amyloid plaque load in the hippocampus. Conversely, PICALM overexpression increased Aβ levels and amyloid plaque load. These data indicate that PICALM, an adaptor protein involved in clathrin-mediated endocytosis, regulates APP internalization and subsequent Aβ generation. PICALM contributes to amyloid plaque load in brain likely via its effect on Aβ metabolism. PMID:22539346

  8. Canine distemper virus persistence in demyelinating encephalitis by swift intracellular cell-to-cell spread in astrocytes is controlled by the viral attachment protein.

    Science.gov (United States)

    Wyss-Fluehmann, Gaby; Zurbriggen, Andreas; Vandevelde, Marc; Plattet, Philippe

    2010-05-01

    The mechanism of viral persistence, the driving force behind the chronic progression of inflammatory demyelination in canine distemper virus (CDV) infection, is associated with non-cytolytic viral cell-to-cell spread. Here, we studied the molecular mechanisms of viral spread of a recombinant fluorescent protein-expressing virulent CDV in primary canine astrocyte cultures. Time-lapse video microscopy documented that CDV spread was very efficient using cell processes contacting remote target cells. Strikingly, CDV transmission to remote cells could occur in less than 6 h, suggesting that a complete viral cycle with production of extracellular free particles was not essential in enabling CDV to spread in glial cells. Titration experiments and electron microscopy confirmed a very low CDV particle production despite higher titers of membrane-associated viruses. Interestingly, confocal laser microscopy and lentivirus transduction indicated expression and functionality of the viral fusion machinery, consisting of the viral fusion (F) and attachment (H) glycoproteins, at the cell surface. Importantly, using a single-cycle infectious recombinant H-knockout, H-complemented virus, we demonstrated that H, and thus potentially the viral fusion complex, was necessary to enable CDV spread. Furthermore, since we could not detect CD150/SLAM expression in brain cells, the presence of a yet non-identified glial receptor for CDV was suggested. Altogether, our findings indicate that persistence in CDV infection results from intracellular cell-to-cell transmission requiring the CDV-H protein. Viral transfer, happening selectively at the tip of astrocytic processes, may help the virus to cover long distances in the astroglial network, "outrunning" the host's immune response in demyelinating plaques, thus continuously eliciting new lesions.

  9. tassel-less1 encodes a boron channel protein required for inflorescence development in maize.

    Science.gov (United States)

    Leonard, April; Holloway, Beth; Guo, Mei; Rupe, Mary; Yu, GongXin; Beatty, Mary; Zastrow-Hayes, Gina; Meeley, Robert; Llaca, Victor; Butler, Karlene; Stefani, Tony; Jaqueth, Jennifer; Li, Bailin

    2014-06-01

    tassel-less1 (tls1) is a classical maize (Zea mays) inflorescence mutant. Homozygous mutant plants have no tassels or very small tassels, and ear development is also impaired. Using a positional cloning approach, ZmNIP3;1 (a NOD26-like intrinsic protein) was identified as the candidate gene for tls1. The ZmNIP3;1 gene is completely deleted in the tls1 mutant genome. Two Mutator-insertional TUSC alleles of ZmNIP3;1 exhibited tls1-like phenotypes, and allelism tests confirmed that the tls1 gene encodes ZmNIP3;1. Transgenic plants with an RNA interference (RNAi) construct to down-regulate ZmNIP3;1 also showed tls1-like phenotypes, further demonstrating that TLS1 is ZmNIP3;1. Sequence analysis suggests that ZmNIP3;1 is a boron channel protein. Foliar application of boron could rescue the tls1 phenotypes and restore the normal tassel and ear development. Gene expression analysis indicated that in comparison with that of the wild type or tls1 plants treated with boron, the transition from the vegetative to reproductive phase or the development of the floral meristem is impaired in the shoot apical meristem of the tls1 mutant plants. It is concluded that the tls1 mutant phenotypes are caused by impaired boron transport, and boron is essential for inflorescence development in maize. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  10. A substrate-fusion protein is trapped inside the Type III Secretion System channel in Shigella flexneri.

    Directory of Open Access Journals (Sweden)

    Kim Dohlich

    2014-01-01

    Full Text Available The Type III Secretion System (T3SS is a macromolecular complex used by Gram-negative bacteria to secrete effector proteins from the cytoplasm across the bacterial envelope in a single step. For many pathogens, the T3SS is an essential virulence factor that enables the bacteria to interact with and manipulate their respective host. A characteristic structural feature of the T3SS is the needle complex (NC. The NC resembles a syringe with a basal body spanning both bacterial membranes and a long needle-like structure that protrudes from the bacterium. Based on the paradigm of a syringe-like mechanism, it is generally assumed that effectors and translocators are unfolded and secreted from the bacterial cytoplasm through the basal body and needle channel. Despite extensive research on T3SS, this hypothesis lacks experimental evidence and the mechanism of secretion is not fully understood. In order to elucidate details of the T3SS secretion mechanism, we generated fusion proteins consisting of a T3SS substrate and a bulky protein containing a knotted motif. Because the knot cannot be unfolded, these fusions are accepted as T3SS substrates but remain inside the NC channel and obstruct the T3SS. To our knowledge, this is the first time substrate fusions have been visualized together with isolated NCs and we demonstrate that substrate proteins are secreted directly through the channel with their N-terminus first. The channel physically encloses the fusion protein and shields it from a protease and chemical modifications. Our results corroborate an elementary understanding of how the T3SS works and provide a powerful tool for in situ-structural investigations in the future. This approach might also be applicable to other protein secretion systems that require unfolding of their substrates prior to secretion.

  11. Enhanced detection of quantum dots labeled protein by simultaneous bismuth electrodeposition into microfluidic channel.

    Science.gov (United States)

    Medina-Sánchez, Mariana; Miserere, Sandrine; Cadevall, Miquell; Merkoçi, Arben

    2016-02-01

    In this study, we propose an electrochemical immunoassay into a disposable microfluidic platform, using quantum dots (QDs) as labels and their enhanced detection using bismuth as an alternative to mercury electrodes. CdSe@ZnS QDs were used to tag human IgG as a model protein and detected through highly sensitive stripping voltammetry of the dissolved metallic component (cadmium in our case). The modification of the screen printed carbon electrodes (SPCEs) was done by a simple electrodeposition of bismuth that was previously mixed with the sample containing QDs. A magneto-immunosandwich assay was performed using a micromixer. A magnet placed at its outlet in order to capture the magnetic beads used as solid support for the immunoassay. SPCEs were integrated at the end of the channel as detector. Different parameters such as bismuth concentration, flow rate, and incubation times, were optimized. The LOD for HIgG in presence of bismuth was 3.5 ng/mL with a RSD of 13.2%. This LOD was about 3.3-fold lower than the one obtained without bismuth. Furthermore, the sensitivity of the system was increased 100-fold respect to experiments carried out with classical screen-printed electrodes, both in presence of bismuth. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Distinct temporal roles for the promyelocytic leukaemia (PML protein in the sequential regulation of intracellular host immunity to HSV-1 infection.

    Directory of Open Access Journals (Sweden)

    Thamir Alandijany

    2018-01-01

    Full Text Available Detection of viral nucleic acids plays a critical role in the induction of intracellular host immune defences. However, the temporal recruitment of immune regulators to infecting viral genomes remains poorly defined due to the technical difficulties associated with low genome copy-number detection. Here we utilize 5-Ethynyl-2'-deoxyuridine (EdU labelling of herpes simplex virus 1 (HSV-1 DNA in combination with click chemistry to examine the sequential recruitment of host immune regulators to infecting viral genomes under low multiplicity of infection conditions. Following viral genome entry into the nucleus, PML-nuclear bodies (PML-NBs rapidly entrapped viral DNA (vDNA leading to a block in viral replication in the absence of the viral PML-NB antagonist ICP0. This pre-existing intrinsic host defence to infection occurred independently of the vDNA pathogen sensor IFI16 (Interferon Gamma Inducible Protein 16 and the induction of interferon stimulated gene (ISG expression, demonstrating that vDNA entry into the nucleus alone is not sufficient to induce a robust innate immune response. Saturation of this pre-existing intrinsic host defence during HSV-1 ICP0-null mutant infection led to the stable recruitment of PML and IFI16 into vDNA complexes associated with ICP4, and led to the induction of ISG expression. This induced innate immune response occurred in a PML-, IFI16-, and Janus-Associated Kinase (JAK-dependent manner and was restricted by phosphonoacetic acid, demonstrating that vDNA polymerase activity is required for the robust induction of ISG expression during HSV-1 infection. Our data identifies dual roles for PML in the sequential regulation of intrinsic and innate immunity to HSV-1 infection that are dependent on viral genome delivery to the nucleus and the onset of vDNA replication, respectively. These intracellular host defences are counteracted by ICP0, which targets PML for degradation from the outset of nuclear infection to promote v

  13. New regulatory mechanisms for the intracellular localization and trafficking of influenza A virus NS1 protein revealed by comparative analysis of A/PR/8/34 and A/Sydney/5/97.

    Science.gov (United States)

    Han, Han; Cui, Zong-Qiang; Wang, Wei; Zhang, Zhi-Ping; Wei, Hong-Ping; Zhou, Ya-Feng; Zhang, Xian-En

    2010-12-01

    During influenza A virus infection, the NS1 protein is engaged in different functions in different intracellular compartments. In this study, we showed that the NS1 of A/PR/8/34 localized in different positions from that of A/Sydney/5/97 when transiently expressed in Madin-Darby canine kidney cells. Residue 221 of NS1 was identified to be a new key residue involved in the C-terminal nuclear localization signal (NLS) and nucleolar localization signal (NoLS) of NS1 from A/Sydney/5/97. Analysis of chimeric NS1 and further mutants showed that residues responsible for the binding between NS1 and the cleavage and polyadenylation specificity factor (CPSF) are correlated with the intracellular localization of transiently expressed NS1 proteins. Fluorescence loss in photobleaching imaging revealed that the NS1 protein with both functional NLSs and nuclear export signal (NES) was able to shuttle between the nucleus and cytoplasm. Drug inhibition experiments and fluorescence resonance energy transfer analysis suggested that NS1 was exported out of the cell nuclei via a Crm1-independent pathway. Moreover, it is likely that another cytoplasmic localization-related sequence exists in the NS1 protein other than the leucine-rich NES. These findings provide new insights into the mechanism of intracellular localization and trafficking of influenza A virus NS1 protein, which is important for understanding its function.

  14. Low density lipoprotein receptor-related protein 1 (LRP1) modulates N-methyl-D-aspartate (NMDA) receptor-dependent intracellular signaling and NMDA-induced regulation of postsynaptic protein complexes.

    Science.gov (United States)

    Nakajima, Chikako; Kulik, Akos; Frotscher, Michael; Herz, Joachim; Schäfer, Michael; Bock, Hans H; May, Petra

    2013-07-26

    The lipoprotein receptor LRP1 is essential in neurons of the central nervous system, as was revealed by the analysis of conditional Lrp1-deficient mouse models. The molecular basis of its neuronal functions, however, is still incompletely understood. Here we show by immunocytochemistry, electron microscopy, and postsynaptic density preparation that LRP1 is located postsynaptically. Basal and NMDA-induced phosphorylation of the transcription factor cAMP-response element-binding protein (CREB) as well as NMDA target gene transcription are reduced in LRP1-deficient neurons. In control neurons, NMDA promotes γ-secretase-dependent release of the LRP1 intracellular domain (LRP1-ICD). However, pull-down and chromatin immunoprecipitation (ChIP) assays showed no direct interaction between the LRP1-ICD and either CREB or target gene promoters. On the other hand, NMDA-induced degradation of the postsynaptic scaffold protein PSD-95 was impaired in the absence of LRP1, whereas its ubiquitination was increased, indicating that LRP1 influences the composition of postsynaptic protein complexes. Accordingly, NMDA-induced internalization of the AMPA receptor subunit GluA1 was impaired in LRP1-deficient neurons. These results show a role of LRP1 in the regulation and turnover of synaptic proteins, which may contribute to the reduced dendritic branching and to the neurological phenotype observed in the absence of LRP1.

  15. Low Density Lipoprotein Receptor-related Protein 1 (LRP1) Modulates N-Methyl-d-aspartate (NMDA) Receptor-dependent Intracellular Signaling and NMDA-induced Regulation of Postsynaptic Protein Complexes*

    Science.gov (United States)

    Nakajima, Chikako; Kulik, Akos; Frotscher, Michael; Herz, Joachim; Schäfer, Michael; Bock, Hans H.; May, Petra

    2013-01-01

    The lipoprotein receptor LRP1 is essential in neurons of the central nervous system, as was revealed by the analysis of conditional Lrp1-deficient mouse models. The molecular basis of its neuronal functions, however, is still incompletely understood. Here we show by immunocytochemistry, electron microscopy, and postsynaptic density preparation that LRP1 is located postsynaptically. Basal and NMDA-induced phosphorylation of the transcription factor cAMP-response element-binding protein (CREB) as well as NMDA target gene transcription are reduced in LRP1-deficient neurons. In control neurons, NMDA promotes γ-secretase-dependent release of the LRP1 intracellular domain (LRP1-ICD). However, pull-down and chromatin immunoprecipitation (ChIP) assays showed no direct interaction between the LRP1-ICD and either CREB or target gene promoters. On the other hand, NMDA-induced degradation of the postsynaptic scaffold protein PSD-95 was impaired in the absence of LRP1, whereas its ubiquitination was increased, indicating that LRP1 influences the composition of postsynaptic protein complexes. Accordingly, NMDA-induced internalization of the AMPA receptor subunit GluA1 was impaired in LRP1-deficient neurons. These results show a role of LRP1 in the regulation and turnover of synaptic proteins, which may contribute to the reduced dendritic branching and to the neurological phenotype observed in the absence of LRP1. PMID:23760271

  16. Trimethyloxonium modification of batrachotoxin-activated Na channels alters functionally important protein residues.

    OpenAIRE

    Cherbavaz, D B

    1995-01-01

    The extracellular side of single batrachotoxin-activated voltage-dependent Na channels isolated from rat skeletal muscle membranes incorporated into neutral planar lipid bilayers were treated in situ with the carboxyl methylating reagent, trimethyloxonium (TMO). These experiments were designed to determine whether TMO alters Na channel function by a general through-space electrostatic mechanism or by methylating specific carboxyl groups essential to channel function. TMO modification reduced ...

  17. Protein-directed synthesis of Mn-doped ZnS quantum dots: a dual-channel biosensor for two proteins.

    Science.gov (United States)

    Wu, Peng; Zhao, Ting; Tian, Yunfei; Wu, Lan; Hou, Xiandeng

    2013-06-03

    Proteins typically have nanoscale dimensions and multiple binding sites with inorganic ions, which facilitates the templated synthesis of nanoparticles to yield nanoparticle-protein hybrids with tailored functionality, water solubility, and tunable frameworks with well-defined structure. In this work, we report a protein-templated synthesis of Mn-doped ZnS quantum dots (QDs) by exploring bovine serum albumin (BSA) as the template. The obtained Mn-doped ZnS QDs give phosphorescence emission centered at 590 nm, with a decay time of about 1.9 ms. A dual-channel sensing system for two different proteins was developed through integration of the optical responses (phosphorescence emission and resonant light scattering (RLS)) of Mn-doped ZnS QDs and recognition of them by surface BSA phosphorescent sensing of trypsin and RLS sensing of lysozyme. Trypsin can digest BSA and remove BSA from the surface of Mn-doped ZnS QDs, thus quenching the phosphorescence of QDs, whereas lysozyme can assemble with BSA to lead to aggregation of QDs and enhanced RLS intensity. The detection limits for trypsin and lysozyme were 40 and 3 nM, respectively. The selectivity of the respective channel for trypsin and lysozyme was evaluated with a series of other proteins. Unlike other protein sensors based on nanobioconjugates, the proposed dual-channel sensor employs only one type of QDs but can detect two different proteins. Further, we found the RLS of QDs can also be useful for studying the BSA-lysozyme binding stoichiometry, which has not been reported in the literature. These successful biosensor applications clearly demonstrate that BSA not only serves as a template for growth of Mn-doped ZnS QDs, but also impacts the QDs for selective recognition of analyte proteins. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Potent neutralization of influenza A virus by a single-domain antibody blocking M2 ion channel protein.

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    Guowei Wei

    Full Text Available Influenza A virus poses serious health threat to humans. Neutralizing antibodies against the highly conserved M2 ion channel is thought to offer broad protection against influenza A viruses. Here, we screened synthetic Camel single-domain antibody (VHH libraries against native M2 ion channel protein. One of the isolated VHHs, M2-7A, specifically bound to M2-expressed cell membrane as well as influenza A virion, inhibited replication of both amantadine-sensitive and resistant influenza A viruses in vitro, and protected mice from a lethal influenza virus challenge. Moreover, M2-7A showed blocking activity for proton influx through M2 ion channel. These pieces of evidence collectively demonstrate for the first time that a neutralizing antibody against M2 with broad specificity is achievable, and M2-7A may have potential for cross protection against a number of variants and subtypes of influenza A viruses.

  19. Cholesterol up-regulates neuronal G protein-gated inwardly rectifying potassium (GIRK) channel activity in the hippocampus.

    Science.gov (United States)

    Bukiya, Anna N; Durdagi, Serdar; Noskov, Sergei; Rosenhouse-Dantsker, Avia

    2017-04-14

    Hypercholesterolemia is a well known risk factor for the development of neurodegenerative disease. However, the underlying mechanisms are mostly unknown. In recent years, it has become increasingly evident that cholesterol-driven effects on physiology and pathophysiology derive from its ability to alter the function of a variety of membrane proteins including ion channels. Yet, the effect of cholesterol on G protein-gated inwardly rectifying potassium (GIRK) channels expressed in the brain is unknown. GIRK channels mediate the actions of inhibitory brain neurotransmitters. As a result, loss of GIRK function can enhance neuron excitability, whereas gain of GIRK function can reduce neuronal activity. Here we show that in rats on a high-cholesterol diet, cholesterol levels in hippocampal neurons are increased. We also demonstrate that cholesterol plays a critical role in modulating neuronal GIRK currents. Specifically, cholesterol enrichment of rat hippocampal neurons resulted in enhanced channel activity. In accordance, elevated currents upon cholesterol enrichment were also observed in Xenopus oocytes expressing GIRK2 channels, the primary GIRK subunit expressed in the brain. Furthermore, using planar lipid bilayers, we show that although cholesterol did not affect the unitary conductance of GIRK2, it significantly enhanced the frequency of channel openings. Last, combining computational and functional approaches, we identified two putative cholesterol-binding sites in the transmembrane domain of GIRK2. These findings establish that cholesterol plays a critical role in modulating GIRK activity in the brain. Because up-regulation of GIRK function can reduce neuronal activity, our findings may lead to novel approaches for prevention and therapy of cholesterol-driven neurodegenerative disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Protein Kinase Cα and P-Type Ca2+ Channel CaV2.1 in Red Blood Cell Calcium Signalling

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    Lisa Wagner-Britz

    2013-06-01

    Full Text Available Background/Aims: Protein kinase Cα (PKCα is activated by an increase in cytosolic Ca2+ in red blood cells (RBCs. Previous work has suggested that PKCα directly stimulates the CaV2.1 channel, whereas other studies revealed that CaV2.1 is insensitive to activation by PKC. The aim of this study was to resolve this discrepancy. Methods: We performed experiments based on a single cell read-out of the intracellular Ca2+ concentration in terms of Fluo-4 fluorescence intensity and phosphatidylserine exposure to the external membrane leaflet. Measurement modalities included flow cytometry and live cell imaging. Results: Treatment of RBCs with phorbol 12-myristate 13-acetate (PMA led to two distinct populations of cells with an increase in intracellular Ca2+: a weak-responding and a strong-responding population. The EC50 of PMA for the number of cells with Ca2+ elevation was 2.7±1.2 µM; for phosphatidylserine exposure to the external membrane surface, it was 2.8±0.5 µM; and for RBC haemolysis, it was 2.9±0.5 µM. Using pharmacological manipulation with the CaV2.1 inhibitor ω-agatoxin TK and the broad protein kinase C inhibitor Gö6983, we are able to show that there are two independent PMA-activated Ca2+ entry processes: the first is independent of CaV2.1 and directly PKCα-activated, while the second is associated with a likely indirect activation of CaV2.1. Further studies using lysophosphatidic acid (LPA as a stimulation agent have provided additional evidence that PKCα and CaV2.1 are not directly interconnected in a signalling chain. Conclusion: Although we provide evidence for a lack of interaction between PKCα and CaV2.1 in RBCs, further studies are required to decipher the signalling relationship between LPA, PKCα and CaV2.1.

  1. Proton transport in a membrane protein channel: two-dimensional infrared spectrum modeling.

    NARCIS (Netherlands)

    Liang, C.; Knoester, J.; Jansen, T.L.Th.A.

    2012-01-01

    We model the two-dimensional infrared (2DIR) spectrum of a proton channel to investigate its applicability as a spectroscopy tool to study the proton transport process in biological systems. Proton transport processes in proton channels are involved in numerous fundamental biochemical reactions.

  2. Protein kinase D regulates the human cardiac L-type voltage-gated calcium channel through serine 1884.

    Science.gov (United States)

    Aita, Yusuke; Kurebayashi, Nagomi; Hirose, Shigehisa; Maturana, Andrés D

    2011-12-15

    Protein kinase D (PKD) regulates the activity of the L-type calcium channel in rat ventricular cardiomyocytes. However, the functional target residues of PKD on the L-type calcium channel remain to be identified. Our aim was to identify the functional phosphorylation sites of PKD on the human L-type calcium channel. The pore subunit of the human CaV1.2 (hCaV1.2) was stably expressed in HEK293 cells. Both the expression of a dominant-negative mutant of PKD and the mutation of serine 1884 but not serine 1930, putative targets of PKD, strongly reduced L-type calcium currents and single channel activity without affecting the channel's expression at the plasma membrane. Our results suggest that serine 1884 is essential for the regulation of hCaV1.2 by PKD. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  3. Synergistic activation of G protein-gated inwardly rectifying potassium channels by cholesterol and PI(4,5)P2.

    Science.gov (United States)

    Bukiya, Anna N; Rosenhouse-Dantsker, Avia

    2017-07-01

    G-protein gated inwardly rectifying potassium (GIRK or Kir3) channels play a major role in the control of the heart rate, and require the membrane phospholipid phosphatidylinositol-bis-phosphate (PI(4,5)P 2 ) for activation. Recently, we have shown that the activity of the heterotetrameric Kir3.1/Kir3.4 channel that underlies atrial K ACh currents was enhanced by cholesterol. Similarly, the activities of both the Kir3.4 homomer and its active pore mutant Kir3.4* (Kir3.4_S143T) were also enhanced by cholesterol. Here we employ planar lipid bilayers to investigate the crosstalk between PI(4,5)P 2 and cholesterol, and demonstrate that these two lipids act synergistically to activate Kir3.4* currents. Further studies using the Xenopus oocytes heterologous expression system suggest that PI(4,5)P 2 and cholesterol act via distinct binding sites. Whereas PI(4,5)P 2 binds to the cytosolic domain of the channel, the putative binding region of cholesterol is located at the center of the transmembrane domain overlapping the central glycine hinge region of the channel. Together, our data suggest that changes in the levels of two key membrane lipids - cholesterol and PI(4,5)P 2 - could act in concert to provide fine-tuning of Kir3 channel function. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density

    Science.gov (United States)

    Ferron, Laurent; Nieto-Rostro, Manuela; Cassidy, John S.; Dolphin, Annette C.

    2014-04-01

    Fragile X syndrome (FXS), the most common heritable form of mental retardation, is characterized by synaptic dysfunction. Synaptic transmission depends critically on presynaptic calcium entry via voltage-gated calcium (CaV) channels. Here we show that the functional expression of neuronal N-type CaV channels (CaV2.2) is regulated by fragile X mental retardation protein (FMRP). We find that FMRP knockdown in dorsal root ganglion neurons increases CaV channel density in somata and in presynaptic terminals. We then show that FMRP controls CaV2.2 surface expression by targeting the channels to the proteasome for degradation. The interaction between FMRP and CaV2.2 occurs between the carboxy-terminal domain of FMRP and domains of CaV2.2 known to interact with the neurotransmitter release machinery. Finally, we show that FMRP controls synaptic exocytosis via CaV2.2 channels. Our data indicate that FMRP is a potent regulator of presynaptic activity, and its loss is likely to contribute to synaptic dysfunction in FXS.

  5. PARAQUAT RESISTANT1, a Golgi-Localized Putative Transporter Protein, Is Involved in Intracellular Transport of Paraquat1[C][W

    Science.gov (United States)

    Li, Jianyong; Mu, Jinye; Bai, Jiaoteng; Fu, Fuyou; Zou, Tingting; An, Fengying; Zhang, Jian; Jing, Hongwei; Wang, Qing; Li, Zhen; Yang, Shuhua; Zuo, Jianru

    2013-01-01

    Paraquat is one of the most widely used herbicides worldwide. In green plants, paraquat targets the chloroplast by transferring electrons from photosystem I to molecular oxygen to generate toxic reactive oxygen species, which efficiently induce membrane damage and cell death. A number of paraquat-resistant biotypes of weeds and Arabidopsis (Arabidopsis thaliana) mutants have been identified. The herbicide resistance in Arabidopsis is partly attributed to a reduced uptake of paraquat through plasma membrane-localized transporters. However, the biochemical mechanism of paraquat resistance remains poorly understood. Here, we report the identification and characterization of an Arabidopsis paraquat resistant1 (par1) mutant that shows strong resistance to the herbicide without detectable developmental abnormalities. PAR1 encodes a putative l-type amino acid transporter protein localized to the Golgi apparatus. Compared with the wild-type plants, the par1 mutant plants show similar efficiency of paraquat uptake, suggesting that PAR1 is not directly responsible for the intercellular uptake of paraquat. However, the par1 mutation caused a reduction in the accumulation of paraquat in the chloroplast, suggesting that PAR1 is involved in the intracellular transport of paraquat into the chloroplast. We identified a PAR1-like gene, OsPAR1, in rice (Oryza sativa). Whereas the overexpression of OsPAR1 resulted in hypersensitivity to paraquat, the knockdown of its expression using RNA interference conferred paraquat resistance on the transgenic rice plants. These findings reveal a unique mechanism by which paraquat is actively transported into the chloroplast and also provide a practical approach for genetic manipulations of paraquat resistance in crops. PMID:23471133

  6. Modulation of Trehalose Dimycolate and Immune System by Rv0774c Protein Enhanced the Intracellular Survival of Mycobacterium smegmatis in Human Macrophages Cell Line

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    Arbind Kumar

    2017-06-01

    Full Text Available Mycobacterium tuberculosis Rv0774c protein was reported previously to express under stress conditions. Therefore, Rv0774c gene was cloned and expressed in Mycobacterium smegmatis, a surrogate host, to determine its role in bacterial persistence and immune modulation in natural environment. The bacterial colonies expressing Rv0774c (Ms_rv0774c were larger, smoother, more moist, and flatter than the control ones (Ms_ve. Enhanced survival of Ms_rv0774c after treatment with streptomycin was observed when compared with control. The cell envelope of Ms_rv0774c was demonstrated to have more trehalose di-mycolate (TDM and lesser amount of mycolylmannosylphosphorylheptaprenol (Myc-PL in comparison to control. Higher intracellular survival rate was observed for Ms_rv0774c as compared to Ms_ve in the THP-1 cells. This could be correlated to the reduction in the levels of reactive NO and iNOS expression. Infection of macrophages with Ms_rv0774c resulted in significantly increased expression of TLR2 receptor and IL-10 cytokines. However, it lowered the production of pro-inflammatory cytokines such as IL-12, TNF-α, IFN-γ, and MCP-1 in Ms_rv0774c infected macrophages in comparison to the control and could be associated with decreased phosphorylation of p38 MAPK. Though, predicted with high antigenicity index bioinformatically, extracellular in nature and accessible to host milieu, Rv0774c was not able to generate humoral response in patient samples. Overall, the present findings indicated that Rv0774c altered the morphology and streptomycin sensitivity by altering the lipid composition of M. smegmatis as well as modulated the immune response in favor of bacterial persistence.

  7. Water channel proteins in the inner ear and their link to hearing impairment and deafness.

    Science.gov (United States)

    Eckhard, Andreas; Gleiser, Corinna; Arnold, Heinz; Rask-Andersen, Helge; Kumagami, Hidetaka; Müller, Marcus; Hirt, Bernhard; Löwenheim, Hubert

    2012-01-01

    The inner ear is a fluid-filled sensory organ that transforms mechanical stimuli into the senses of hearing and balance. These neurosensory functions depend on the strict regulation of the volume of the two major extracellular fluid domains of the inner ear, the perilymph and the endolymph. Water channel proteins, or aquaporins (AQPs), are molecular candidates for the precise regulation of perilymph and endolymph volume. Eight AQP subtypes have been identified in the membranous labyrinth of the inner ear. Similar AQP subtypes are also expressed in the kidney, where they function in whole-body water regulation. In the inner ear, AQP subtypes are ubiquitously expressed in distinct cell types, suggesting that AQPs have an important physiological role in the volume regulation of perilymph and endolymph. Furthermore, disturbed AQP function may have pathophysiological relevance and may turn AQPs into therapeutic targets for the treatment of inner ear diseases. In this review, we present the currently available knowledge regarding the expression and function of AQPs in the inner ear. We give special consideration to AQP subtypes AQP2, AQP4 and AQP5, which have been studied most extensively. The potential functions of AQP2 and AQP5 in the resorption and secretion of endolymph and of AQP4 in the equilibration of cell volume are described. The pathophysiological implications of these AQP subtypes for inner ear diseases, that appear to involve impaired fluid regulation, such as Menière's disease and Sjögren's syndrome, are discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Negative inotropic effect of carbachol and interaction between acetylcholine receptor-operated potassium channel (K.ACh channel) and GTP binding protein in mouse isolated atrium--a novel methodological trial.

    Science.gov (United States)

    Okada, Muneyoshi; Noma, Chihiro; Yamawaki, Hideyuki; Hara, Yukio

    2013-01-01

    Interaction between acetylcholine receptor-operated potassium channel (K.ACh channel) and GTP binding protein was examined by an immunoprecipitation-Western blotting system in mouse isolated atrium. The carbachol-induced negative inotropic action in indomethacin-pretreated mouse atrium was significantly inhibited by a K.ACh channel blocker, tertiapin or atropine. Kir3.1 K.ACh channel (Kir3.1) was immunoprecipitated with a mouse anti-Kir3.1 antibody. Coprecipitating Gβ with Kir3.1, detected by Western blotting, was significantly augmented by carbachol. Atropine, but not tertiapin, significantly inhibited the carbachol-induced coprecipitating Gβ with Kir3.1. The data indicate that immunoprecipitation with Kir3.1 and Western blotting of Gβ system is a useful method for assessing interaction between K.ACh channel and GTP binding protein in mouse atrium.

  9. SPAK and OSR1 Sensitive Cell Membrane Protein Abundance and Activity of KCNQ1/E1 K+ Channels

    Directory of Open Access Journals (Sweden)

    Bernat Elvira

    2015-11-01

    Full Text Available Background/Aims: KCNQ1/E1 channels are expressed in diverse tissues and serve a variety of functions including endolymph secretion in the inner ear, cardiac repolarization, epithelial transport and cell volume regulation. Kinases involved in regulation of epithelial transport and cell volume include SPAK (SPS1-related proline/alanine-rich kinase and OSR1 (oxidative stress-responsive kinase 1, which are under control of WNK (with-no-K[Lys] kinases. The present study explored whether KCNQ1/E1 channels are regulated by SPAK and/or OSR1. Methods: cRNA encoding KCNQ1/E1 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active T233ESPAK, WNK insensitive T233ASPAK, catalytically inactive D212ASPAK, wild-type OSR1, constitutively active T185EOSR1, WNK insensitive T185AOSR1 and catalytically inactive D164AOSR1. Voltage gated K+ channel activity was quantified utilizing dual electrode voltage clamp and KCNQ1/E1 channel protein abundance in the cell membrane utilizing chemiluminescence of KCNQ1/E1 containing an extracellular Flag tag epitope (KCNQ1-Flag/E1. Results: KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly enhanced by wild-type SPAK and T233ESPAK, but not by T233ASPAK and D212ASPAK. Similarly, KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly increased by wild-type OSR1 and T185EOSR1, but not by T185AOSR1 and D164AOSR1. Conclusions: SPAK and OSR1 participate in the regulation of KCNQ1/E1 protein abundance and activity.

  10. SPAK and OSR1 Sensitive Cell Membrane Protein Abundance and Activity of KCNQ1/E1 K+ Channels.

    Science.gov (United States)

    Elvira, Bernat; Warsi, Jamshed; Fezai, Myriam; Munoz, Carlos; Lang, Florian

    2015-01-01

    KCNQ1/E1 channels are expressed in diverse tissues and serve a variety of functions including endolymph secretion in the inner ear, cardiac repolarization, epithelial transport and cell volume regulation. Kinases involved in regulation of epithelial transport and cell volume include SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), which are under control of WNK (with-no-K[Lys]) kinases. The present study explored whether KCNQ1/E1 channels are regulated by SPAK and/or OSR1. cRNA encoding KCNQ1/E1 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active T233ESPAK, WNK insensitive T233ASPAK, catalytically inactive D212ASPAK, wild-type OSR1, constitutively active T185EOSR1, WNK insensitive T185AOSR1 and catalytically inactive D164AOSR1. Voltage gated K+ channel activity was quantified utilizing dual electrode voltage clamp and KCNQ1/E1 channel protein abundance in the cell membrane utilizing chemiluminescence of KCNQ1/E1 containing an extracellular Flag tag epitope (KCNQ1-Flag/E1). KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly enhanced by wild-type SPAK and T233ESPAK, but not by T233ASPAK and D212ASPAK. Similarly, KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly increased by wild-type OSR1 and T185EOSR1, but not by T185AOSR1 and D164AOSR1. SPAK and OSR1 participate in the regulation of KCNQ1/E1 protein abundance and activity. © 2015 S. Karger AG, Basel.

  11. Expanding the neuron's calcium signaling repertoire: intracellular calcium release via voltage-induced PLC and IP3R activation.

    Directory of Open Access Journals (Sweden)

    Stefanie Ryglewski

    2007-04-01

    Full Text Available Neuronal calcium acts as a charge carrier during information processing and as a ubiquitous intracellular messenger. Calcium signals are fundamental to numerous aspects of neuronal development and plasticity. Specific and independent regulation of these vital cellular processes is achieved by a rich bouquet of different calcium signaling mechanisms within the neuron, which either can operate independently or may act in concert. This study demonstrates the existence of a novel calcium signaling mechanism by simultaneous patch clamping and calcium imaging from acutely isolated central neurons. These neurons possess a membrane voltage sensor that, independent of calcium influx, causes G-protein activation, which subsequently leads to calcium release from intracellular stores via phospholipase C and inositol 1,4,5-trisphosphate receptor activation. This allows neurons to monitor activity by intracellular calcium release without relying on calcium as the input signal and opens up new insights into intracellular signaling, developmental regulation, and information processing in neuronal compartments lacking calcium channels.

  12. Protein structure and ionic selectivity in calcium channels: Selectivity filter size, not shape, matters

    OpenAIRE

    Malasics, Attila; Gillespie, Dirk; Nonner, Wolfgang; Henderson, Douglas; Eisenberg, Bob; Boda, Dezső

    2009-01-01

    Calcium channels have highly charged selectivity filters (4 COO− groups) that attract cations in to balance this charge and minimize free energy, forcing the cations (Na+ and Ca2+) to compete for space in the filter. A reduced model was developed to better understand the mechanism of ion selectivity in calcium channels. The charge/space competition (CSC) mechanism implies that Ca2+ is more efficient in balancing the charge of the filter because it provides twice the charge as Na+ while occupy...

  13. A guanine nucleotide exchange factor for Rab5 proteins is essential for intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm.

    Science.gov (United States)

    Fukuda, Masako; Wen, Liuying; Satoh-Cruz, Mio; Kawagoe, Yasushi; Nagamura, Yoshiaki; Okita, Thomas W; Washida, Haruhiko; Sugino, Aya; Ishino, Sonoko; Ishino, Yoshizumi; Ogawa, Masahiro; Sunada, Mariko; Ueda, Takashi; Kumamaru, Toshihiro

    2013-06-01

    Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as a precursor, which are then transported via the Golgi to protein storage vacuoles (PSVs), where they are proteolytically processed into acidic and basic subunits. The glutelin precursor mutant6 (glup6) accumulates abnormally large amounts of proglutelin. Map-base cloning studies showed that glup6 was a loss-of-function mutant of guanine nucleotide exchange factor (GEF), which activates Rab GTPase, a key regulator of membrane trafficking. Immunofluorescence studies showed that the transport of proglutelins and α-globulins to PSV was disrupted in glup6 endosperm. Secreted granules of glutelin and α-globulin were readily observed in young glup6 endosperm, followed by the formation of large dilated paramural bodies (PMBs) containing both proteins as the endosperm matures. The PMBs also contained membrane biomarkers for the Golgi and prevacuolar compartment as well as the cell wall component, β-glucan. Direct evidence was gathered showing that GLUP6/GEF activated in vitro GLUP4/Rab5 as well as several Arabidopsis (Arabidopsis thaliana) Rab5 isoforms to the GTP-bound form. Therefore, loss-of-function mutations in GEF or Rab5 disrupt the normal transport of proglutelin from the Golgi to PSVs, resulting in the initial extracellular secretion of these proteins followed, in turn, by the formation of PMBs. Overall, our results indicate that GLUP6/GEF is the activator of Rab5 GTPase and that the cycling of GTP- and GDP-bound forms of this regulatory protein is essential for the intracellular transport of proglutelin and α-globulin from the Golgi to PSVs and in the maintenance of the general structural organization of the endomembrane system in rice seeds.

  14. Regulator of G-protein signalling and GoLoco proteins suppress TRPC4 channel function via acting at Gαi/o.

    Science.gov (United States)

    Jeon, Jae-Pyo; Thakur, Dhananjay P; Tian, Jin-Bin; So, Insuk; Zhu, Michael X

    2016-05-15

    Transient receptor potential canonical 4 (TRPC4) forms non-selective cation channels implicated in the regulation of diverse physiological functions. Previously, TRPC4 was shown to be activated by the Gi/o subgroup of heterotrimeric G-proteins involving Gαi/o, rather than Gβγ, subunits. Because the lifetime and availability of Gα-GTP are regulated by regulators of G-protein signalling (RGS) and Gαi/o-Loco (GoLoco) domain-containing proteins via their GTPase-activating protein (GAP) and guanine-nucleotide-dissociation inhibitor (GDI) functions respectively, we tested how RGS and GoLoco domain proteins affect TRPC4 currents activated via Gi/o-coupled receptors. Using whole-cell patch-clamp recordings, we show that both RGS and GoLoco proteins [RGS4, RGS6, RGS12, RGS14, LGN or activator of G-protein signalling 3 (AGS3)] suppress receptor-mediated TRPC4 activation without causing detectable basal current or altering surface expression of the channel protein. The inhibitory effects are dependent on the GAP and GoLoco domains and facilitated by enhancing membrane targeting of the GoLoco protein AGS3. In addition, RGS, but not GoLoco, proteins accelerate desensitization of receptor-activation evoked TRPC4 currents. The inhibitory effects of RGS and GoLoco domains are additive and are most prominent with RGS12 and RGS14, which contain both RGS and GoLoco domains. Our data support the notion that the Gα, but not Gβγ, arm of the Gi/o signalling is involved in TRPC4 activation and unveil new roles for RGS and GoLoco domain proteins in fine-tuning TRPC4 activities. The versatile and diverse functions of RGS and GoLoco proteins in regulating G-protein signalling may underlie the complexity of receptor-operated TRPC4 activation in various cell types under different conditions. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  15. Putative new groups of invertebrate water channels based on the snail Helix pomatia L. (Helicidae) MIP protein identification and phylogenetic analysis.

    Science.gov (United States)

    Kosicka, Ewa; Grobys, Daria; Kmita, Hanna; Lesicki, Andrzej; Pieńkowska, Joanna R

    2016-12-01

    Water channel proteins, classified as a family of Membrane Intrinsic Proteins (MIPs) superfamily, enable rapid movement of water and small uncharged molecules through biological membranes. Although water channel proteins are required in several important processes characteristic for the animals, such as osmoregulation, mucus secretion, or defense against desiccation, molluscs, until now, have been very poorly explored in this aspect. Therefore, we decided to study MIPs in Helix pomatia L. applied as a model in studies on terrestrial snail physiology. Our studies consisted in: the snail organ transcriptome sequencing and consecutive bioinformatic analysis of the predicted protein, estimation of the encoding transcript expression (qPCR), investigation of the predicted protein function in the yeast Saccharomyces cerevisiae cells, and the phylogenetic analysis. We identified six water channel proteins, named HpAQP1 to HpAQP6. All of them were proven to transport water, two of them (HpAQP3 and HpAQP4) were also shown to be able to transport glycerol, and other two (HpAQP5 and HpAQP6) to transport H 2 O 2 . Phylogenetic analysis indicated that the proteins either fell into aquaporins (HpAQP1, HpAQP2 and HpAQP5) or formed new groups of invertebrate water channel proteins, not described until now, that we suggest to term malacoglyceroporins (HpAQP3 and HpAQP4) and malacoaquaporins (HpAQP6). Thus, the classification of animal water channels based on the vertebrate proteins and including aquaporin, aquaglyceroporin, S-aquaporin and AQP8-type grades does not reflect diversity of these proteins in invertebrates. The obtained results provide important data concerning diversity of water channel protein repertoire in aquatic and terrestrial invertebrates and should also contribute to the improvement of animal water channel classification system. Copyright © 2016 Elsevier GmbH. All rights reserved.

  16. Brain-derived neurotrophic factor modulation of Kv1.3 channel is disregulated by adaptor proteins Grb10 and nShc

    Directory of Open Access Journals (Sweden)

    Marks David R

    2009-01-01

    Full Text Available Abstract Background Neurotrophins are important regulators of growth and regeneration, and acutely, they can modulate the activity of voltage-gated ion channels. Previously we have shown that acute brain-derived neurotrophic factor (BDNF activation of neurotrophin receptor tyrosine kinase B (TrkB suppresses the Shaker voltage-gated potassium channel (Kv1.3 via phosphorylation of multiple tyrosine residues in the N and C terminal aspects of the channel protein. It is not known how adaptor proteins, which lack catalytic activity, but interact with members of the neurotrophic signaling pathway, might scaffold with ion channels or modulate channel activity. Results We report the co-localization of two adaptor proteins, neuronal Src homology and collagen (nShc and growth factor receptor-binding protein 10 (Grb10, with Kv1.3 channel as demonstrated through immunocytochemical approaches in the olfactory bulb (OB neural lamina. To further explore the specificity and functional ramification of adaptor/channel co-localization, we performed immunoprecipitation and Western analysis of channel, kinase, and adaptor transfected human embryonic kidney 293 cells (HEK 293. nShc formed a direct protein-protein interaction with Kv1.3 that was independent of BDNF-induced phosphorylation of Kv1.3, whereas Grb10 did not complex with Kv1.3 in HEK 293 cells. Both adaptors, however, co-immunoprecipitated with Kv1.3 in native OB. Grb10 was interestingly able to decrease the total expression of Kv1.3, particularly at the membrane surface, and subsequently eliminated the BDNF-induced phosphorylation of Kv1.3. To examine the possibility that the Src homology 2 (SH2 domains of Grb10 were directly binding to basally phosphorylated tyrosines in Kv1.3, we utilized point mutations to substitute multiple tyrosine residues with phenylalanine. Removal of the tyrosines 111–113 and 449 prevented Grb10 from decreasing Kv1.3 expression. In the absence of either adaptor protein

  17. Biosynthesis of gold nanoparticles assisted by the intracellular protein extract of Pycnoporus sanguineus and its catalysis in degradation of 4-nitroaniline

    Science.gov (United States)

    Shi, Chaohong; Zhu, Nengwu; Cao, Yanlan; Wu, Pingxiao

    2015-03-01

    The development of green procedure for the synthesis of gold nanoparticles (AuNPs) has gained great interest in the field of nanotechnology. Biological synthetic routes are considered to be environmentally benign and cost-effective. In the present study, the feasibility of AuNPs' synthesis via intracellular protein extract (IPE) of Pycnoporus sanguineus was explored. The characteristics of generated particles of formation, crystalline nature, and morphology and dimension were analyzed by UV-vis spectroscopy, X-ray diffraction (XRD) and transmission electron microscopy (TEM), respectively. UV-vis spectra exhibited strong absorption peaks in 520 to 560 nm, indicating the formation of AuNPs. XRD analysis revealed that the formed AuNPs were purely crystalline in nature. TEM observation showed that AuNPs with various shapes including spherical, pseudo-spherical, triangular, truncated triangular, pentagonal, and hexagonal, ranging from several to several hundred nanometers, were synthesized under different conditions. The average size of AuNPs decreased from 61.47 to 29.30 nm as the IPE addition increased from 10 to 80 mL. When the initial gold ion concentration changed from 0.5 to 2.0 mM, the average size rose from 25.88 to 51.99 nm. As in the case of solution pH, the average size was 84.29 nm with solution pH of 2.0, which diminished to 6.07 nm with solution pH of 12.0. Fourier transform infrared (FTIR) analysis implied that the functional groups including hydroxyl, amine, and carboxyl were involved in the reduction of gold ions and stabilization of AuNPs. The catalysis results showed that 0.019 mg of AuNPs with average size of 6.07 nm could catalyze the complete degradation of 12.5 μmol of 4-nitroaniline within 6 min and the degradation rate increased drastically with the addition of AuNPs. All the results suggested that the IPE of P. sanguineus could be potentially applied for the eco-friendly synthesis of AuNPs.

  18. Effect of protein supplementation on forage utilization by cattle in annual grass-dominated rangelands in the Channel Scablands of Eastern Washington

    Science.gov (United States)

    Medusahead (Taeniatherum caput-medusae [L.] Nevski) has become a major component replacing vegetation on the Channeled Scablands of eastern Washington. Livestock typically avoid grazing medusahead and forage alternatives are becoming limited in the region. We hypothesized that protein supplementat...

  19. A Ca-activated K channel from rabbit renal brush-border membrane vesicles in planar lipid bilayers.

    Science.gov (United States)

    Zweifach, A; Desir, G V; Aronson, P S; Giebisch, G H

    1991-07-01

    Rabbit renal brush-border membranes were fused to planar lipid bilayers to gain insight into the nature and properties of ion channels from the luminal membrane of the proximal tubule. Fusion was obtained using osmotic gradients. A large conductance channel was commonly observed. Measurements of reversal potentials indicated that the channel was selective for K over Rb, Na, and Cl. Channel open probability was increased by membrane depolarization and by increased Ca activity on the intracellular face of the channel. The channel was inhibited by charybdotoxin (CTX), a protein from leiurus venom, from the external side of the channel. The channel was also blocked by Ba and quinidine added to the intracellular bathing solution. Na added to the intracellular bathing solution reduced current amplitude in a voltage-dependent fashion. In addition, methylisobutyl amiloride, an analogue of the K-sparing diuretic amiloride, inhibited channel activity when added to the external solution. The possible physiological role of the channel is discussed. The usefulness to the study of renal ion channels of the technique of fusing membrane vesicles to planar lipid bilayers is evaluated.

  20. Use of magnetic nanobeads to study intracellular antigen processing

    Energy Technology Data Exchange (ETDEWEB)

    Perrin-Cocon, Laure A.; Chesne, Serge; Pignot-Paintrand, Isabelle; Marche, Patrice N.; Villiers, Christian L. E-mail: christian.villiers@cea.fr

    2001-07-01

    Magnetic nanobeads were covalently linked to antigens and used as a tool to simultaneously follow their intracellular transport into the cells and specifically purify the intracellular compartments implicated in antigen processing. The protein content of these vesicles was analysed by 2D-electrophoresis. Furthermore, nanobeads allowed intracellular localisation of the antigen in electron and fluorescence microscopy.

  1. Use of magnetic nanobeads to study intracellular antigen processing

    International Nuclear Information System (INIS)

    Perrin-Cocon, Laure A.; Chesne, Serge; Pignot-Paintrand, Isabelle; Marche, Patrice N.; Villiers, Christian L.

    2001-01-01

    Magnetic nanobeads were covalently linked to antigens and used as a tool to simultaneously follow their intracellular transport into the cells and specifically purify the intracellular compartments implicated in antigen processing. The protein content of these vesicles was analysed by 2D-electrophoresis. Furthermore, nanobeads allowed intracellular localisation of the antigen in electron and fluorescence microscopy

  2. Cav1.3 channel α1D protein is overexpressed and modulates androgen receptor transactivation in prostate cancers.

    Science.gov (United States)

    Chen, Ruibao; Zeng, Xing; Zhang, Ruitao; Huang, Jiaoti; Kuang, Xiangxing; Yang, Jun; Liu, Jihong; Tawfik, Ossama; Thrasher, James Brantley; Li, Benyi

    2014-07-01

    Widespread use of L-type calcium channel blockers for treating hypertension has led to multiple epidemiologic studies to assess the risk of prostate cancer incidence. These studies revealed a reverse correlation between the likelihood of prostate cancer risk and the use of L-type calcium channel blockers among men without family history but the mechanism was not clear. In this study, we examined the expression profiles of multiple L-type calcium channel genes in prostate cancers and determined their functional roles in androgen receptor (AR) transactivation and cell growth. By reanalyzing the ONCOMINE database, we found that L-type calcium channel CACNA1D gene expression levels in cancer tissues were significantly higher than noncancer tissues in 14 of 15 published complementary deoxyribonucleic acid microarray data sets, of which 9 data sets showed an increase of 2- to 17-folds. Quantitative polymerase chain reaction and immunostaining experiments revealed that CACNA1D gene and its coding protein α1D were highly expressed in prostate cancers, especially in castration-resistant diseases, compared with benign prostate tissues. Consistent with the notion of CACNA1D as an ERG-regulated gene, CACNA1D gene expression levels were significantly higher in prostate cancers with TMPRSS2-ERG gene fusion compared with the cases without this gene fusion. Blocking L-type channel's function or knocking down CACNA1D gene expression significantly suppressed androgen-stimulated Ca(2+) influx, AR transactivation, and cell growth in prostate cancer cells. Taken together, these data suggest that CACNA1D gene overexpression is associated with prostate cancer progression and might play an important role in Ca(2+) influx, AR activation, and cell growth in prostate cancer cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Structure-function of proteins interacting with the α1 pore-forming subunit of high-voltage-activated calcium channels

    Science.gov (United States)

    Neely, Alan; Hidalgo, Patricia

    2014-01-01

    Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1) associated with four additional polypeptide chains β, α2, δ, and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels. PMID:24917826

  4. Structure-function of proteins interacting with the alpha1 pore-forming subunit of high voltage-activated calcium channel

    Directory of Open Access Journals (Sweden)

    Alan eNeely

    2014-06-01

    Full Text Available Openings of high-voltage-activated calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, high-voltage-activated calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1 associated with four additional polypeptide chains β, α2, δ and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of high-voltage-activated calcium channels.

  5. Optochemokine Tandem for Light-Control of Intracellular Ca2.

    Directory of Open Access Journals (Sweden)

    Katrin Feldbauer

    Full Text Available An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca2+-permeable cation channel Channelrhodopsin-2(L132C, CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca2+ by tandem endosomes into the cytosol via CatCh was visualized using the Ca2+-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca2+ in response to light.

  6. Multi-scaled normal mode analysis method for dynamics simulation of protein-membrane complexes: A case study of potassium channel gating motion correlations

    Science.gov (United States)

    Wu, Xiaokun; Han, Min; Ming, Dengming

    2015-10-01

    Membrane proteins play critically important roles in many cellular activities such as ions and small molecule transportation, signal recognition, and transduction. In order to fulfill their functions, these proteins must be placed in different membrane environments and a variety of protein-lipid interactions may affect the behavior of these proteins. One of the key effects of protein-lipid interactions is their ability to change the dynamics status of membrane proteins, thus adjusting their functions. Here, we present a multi-scaled normal mode analysis (mNMA) method to study the dynamics perturbation to the membrane proteins imposed by lipid bi-layer membrane fluctuations. In mNMA, channel proteins are simulated at all-atom level while the membrane is described with a coarse-grained model. mNMA calculations clearly show that channel gating motion can tightly couple with a variety of membrane deformations, including bending and twisting. We then examined bi-channel systems where two channels were separated with different distances. From mNMA calculations, we observed both positive and negative gating correlations between two neighboring channels, and the correlation has a maximum as the channel center-to-center distance is close to 2.5 times of their diameter. This distance is larger than recently found maximum attraction distance between two proteins embedded in membrane which is 1.5 times of the protein size, indicating that membrane fluctuation might impose collective motions among proteins within a larger area. The hybrid resolution feature in mNMA provides atomic dynamics information for key components in the system without costing much computer resource. We expect it to be a conventional simulation tool for ordinary laboratories to study the dynamics of very complicated biological assemblies. The source code is available upon request to the authors.

  7. Pannexin1 channel proteins in the zebrafish retina have shared and unique properties

    NARCIS (Netherlands)

    Kurtenbach, Sarah; Prochnow, Nora; Kurtenbach, Stefan; Klooster, Jan; Zoidl, Christiane; Dermietzel, Rolf; Kamermans, Maarten; Zoidl, Georg

    2013-01-01

    In mammals, a single pannexin1 gene (Panx1) is widely expressed in the CNS including the inner and outer retinae, forming large-pore voltage-gated membrane channels, which are involved in calcium and ATP signaling. Previously, we discovered that zebrafish lack Panx1 expression in the inner retina,

  8. Active zone protein Bassoon co-localizes with presynaptic calcium channel, modifies channel function, and recovers from aging related loss by exercise.

    Science.gov (United States)

    Nishimune, Hiroshi; Numata, Tomohiro; Chen, Jie; Aoki, Yudai; Wang, Yonghong; Starr, Miranda P; Mori, Yasuo; Stanford, John A

    2012-01-01

    The P/Q-type voltage-dependent calcium channels (VDCCs) are essential for synaptic transmission at adult mammalian neuromuscular junctions (NMJs); however, the subsynaptic location of VDCCs relative to active zones in rodent NMJs, and the functional modification of VDCCs by the interaction with active zone protein Bassoon remain unknown. Here, we show that P/Q-type VDCCs distribute in a punctate pattern within the NMJ presynaptic terminals and align in three dimensions with Bassoon. This distribution pattern of P/Q-type VDCCs and Bassoon in NMJs is consistent with our previous study demonstrating the binding of VDCCs and Bassoon. In addition, we now show that the interaction between P/Q-type VDCCs and Bassoon significantly suppressed the inactivation property of P/Q-type VDCCs, suggesting that the Ca(2+) influx may be augmented by Bassoon for efficient synaptic transmission at NMJs. However, presynaptic Bassoon level was significantly attenuated in aged rat NMJs, which suggests an attenuation of VDCC function due to a lack of this interaction between VDCC and Bassoon. Importantly, the decreased Bassoon level in aged NMJs was ameliorated by isometric strength training of muscles for two months. The training increased Bassoon immunoreactivity in NMJs without affecting synapse size. These results demonstrated that the P/Q-type VDCCs preferentially accumulate at NMJ active zones and play essential role in synaptic transmission in conjunction with the active zone protein Bassoon. This molecular mechanism becomes impaired by aging, which suggests altered synaptic function in aged NMJs. However, Bassoon level in aged NMJs can be improved by muscle exercise.

  9. Expression profiling and intracellular localization studies of the novel Proline-, Histidine-, and Glycine-rich protein 1 suggest an essential role in gastro-intestinal epithelium and a potential clinical application in colorectal cancer diagnostics.

    Science.gov (United States)

    Oltedal, Satu; Skaland, Ivar; Maple-Grødem, Jodi; Tjensvoll, Kjersti; Janssen, Emiel A M; Gilje, Bjørnar; Smaaland, Rune; Heikkilä, Reino; Nordgård, Oddmund

    2018-02-07

    The primary function of the intestines is the absorption of water and nutrients. Although our knowledge about these processes on the cellular level is extensive, a number of important intracellular elements remain unknown. Here, we characterize the novel proline-, histidine-, glycine-rich 1 (PHGR1) mRNA and protein on the molecular level and propose a functional role of the PHGR1 protein in the intestinal and gastric epithelium. PHGR1 mRNA and protein expression in human tissues and cell lines were characterized by quantitative RT-PCR, in situ hybridization, Northern blotting, Western blotting, and immunohistochemistry. Glycosylation was assessed by a chemical deglycosylation assay, whereas intracellular localization was studied by immunofluorescent staining of cell line cells. PHGR1 mRNA levels in HT29 cells was reduced by RNA interference and the resulting global changes in gene expression assessed by microarray hybridization. PHGR1 mRNA and protein were found to be expressed specifically in epithelial cells of intestinal mucosa, with the highest expression in the most mature and differentiated cells. PHGR1 protein was found to be glycosylated and to localize to both the cytoplasm and nucleus. Transcript profiling and gene ontology analysis of HT29 cells subjected to PHGR1 knockdown suggested a functional relationship with transport and metabolic processes. Examination of PHGR1 mRNA and protein levels in lymph nodes with known colorectal cancer metastases indicated that they may serve as biomarkers for detection of such metastases. Functional analyses of the novel PHGR1 mRNA and protein suggest an essential role in gastrointestinal epithelium and a clinical application in detection of colorectal cancer lymph node metastases.

  10. Retinal Cyclic Nucleotide-Gated Channels: From Pathophysiology to Therapy

    Directory of Open Access Journals (Sweden)

    Stylianos Michalakis

    2018-03-01

    Full Text Available The first step in vision is the absorption of photons by the photopigments in cone and rod photoreceptors. After initial amplification within the phototransduction cascade the signal is translated into an electrical signal by the action of cyclic nucleotide-gated (CNG channels. CNG channels are ligand-gated ion channels that are activated by the binding of cyclic guanosine monophosphate (cGMP or cyclic adenosine monophosphate (cAMP. Retinal CNG channels transduce changes in intracellular concentrations of cGMP into changes of the membrane potential and the Ca2+ concentration. Structurally, the CNG channels belong to the superfamily of pore-loop cation channels and share a common gross structure with hyperpolarization-activated cyclic nucleotide-gated (HCN channels and voltage-gated potassium channels (KCN. In this review, we provide an overview on the molecular properties of CNG channels and describe their physiological role in the phototransduction pathways. We also discuss insights into the pathophysiological role of CNG channel proteins that have emerged from the analysis of CNG channel-deficient animal models and human CNG channelopathies. Finally, we summarize recent gene therapy activities and provide an outlook for future clinical application.

  11. DNA translocation across protein channels: How does a polymer worm through a hole?

    Science.gov (United States)

    Muthukumar, M.

    2001-03-01

    Free energy barriers control the translocation of polymers through narrow channels. Based on an analogy with the classical nucleation and growth process, we have calculated the translocation time and its dependencies on the length, stiffness, and sequence of the polymer, solution conditions, and the strength of the driving electrochemical potential gradient. Our predictions will be compared with experimental results and prospects of reading polymer sequences.

  12. Investigating Internalization and Intracellular Trafficking of GPCRs

    DEFF Research Database (Denmark)

    Foster, Simon R; Bräuner-Osborne, Hans

    2017-01-01

    for signal transduction. One of the major mechanisms for GPCR regulation involves their endocytic trafficking, which serves to internalize the receptors from the plasma membrane and thereby attenuate G protein-dependent signaling. However, there is accumulating evidence to suggest that GPCRs can signal...... independently of G proteins, as well as from intracellular compartments including endosomes. It is in this context that receptor internalization and intracellular trafficking have attracted renewed interest within the GPCR field. In this chapter, we will review the current understanding and methodologies...... that have been used to investigate internalization and intracellular signaling of GPCRs, with a particular focus on emerging real-time techniques. These recent developments have improved our understanding of the complexities of GPCR internalization and intracellular signaling and suggest that the broader...

  13. Small-conductance Ca2+-activated K+ channels: form and function.

    Science.gov (United States)

    Adelman, John P; Maylie, James; Sah, Pankaj

    2012-01-01

    Small-conductance Ca(2+)-activated K(+) channels (SK channels) are widely expressed throughout the central nervous system. These channels are activated solely by increases in intracellular Ca(2+). SK channels are stable macromolecular complexes of the ion pore-forming subunits with calmodulin, which serves as the intrinsic Ca(2+) gating subunit, as well as with protein kinase CK2 and protein phosphatase 2A, which modulate Ca(2+) sensitivity. Well-known for their roles in regulating somatic excitability in central neurons, SK channels are also expressed in the postsynaptic membrane of glutamatergic synapses, where their activation and regulated trafficking modulate synaptic transmission and the induction and expression of synaptic plasticity, thereby affecting learning and memory. In this review we discuss the molecular and functional properties of SK channels and their physiological roles in central neurons.

  14. Regulation of BK channels by auxiliary γ subunits

    Directory of Open Access Journals (Sweden)

    Jiyuan eZhang

    2014-10-01

    Full Text Available The large-conductance, calcium- and voltage-activated potassium (BK channel has the largest single-channel conductance among potassium channels and can be activated by both membrane depolarization and increases in intracellular calcium concentration. BK channels consist of pore-forming, voltage- and calcium-sensing α subunits, either alone or in association with regulatory subunits. BK channels are widely expressed in various tissues and cells including both excitable and non-excitable cells and display diverse biophysical and pharmacological characteristics. This diversity can be explained in part by posttranslational modifications and alternative splicing of the α subunit, which is encoded by a single gene, KCNMA1, as well as by tissue-specific β subunit modulation. Recently, a leucine-rich repeat-containing membrane protein, LRRC26, was found to interact with BK channels and cause an unprecedented large negative shift (~-140 mV in the voltage dependence of the BK channel activation. LRRC26 allows BK channels to open even at near-physiological calcium concentration and membrane voltage in non-excitable cells. Three LRRC26-related proteins, LRRC52, LRRC55, and LRRC38, were subsequently identified as BK channel modulators. These LRRC proteins are structurally and functionally distinct from the BK channel β subunits and were designated as γ subunits. The discovery of the γ subunits adds a new dimension to BK channel regulation and improves our understanding of the physiological functions of BK channels in various tissues and cell types. Unlike BK channel β subunits, which have been intensively investigated both mechanistically and physiologically, our understanding of the γ subunits is very limited at this stage. This article reviews the structure, modulatory mechanisms, physiological relevance, and potential therapeutic implications of γ subunits as they are currently understood.

  15. Effect of insecticidal fusion proteins containing spider toxins targeting sodium and calcium ion channels on pyrethroid-resistant strains of peach-potato aphid (Myzus persicae).

    Science.gov (United States)

    Yang, Sheng; Fitches, Elaine; Pyati, Prashant; Gatehouse, John A

    2015-07-01

    The recombinant fusion proteins Pl1a/GNA and Hv1a/GNA contain the spider venom peptides δ-amaurobitoxin-PI1a or ω-hexatoxin-Hv1a respectively, linked to snowdrop lectin (GNA). Pl1a targets receptor site 4 of insect voltage-gated sodium channels (NaCh), while Hv1a targets voltage-gated calcium channels. Insecticide-resistant strains of peach-potato aphid (Myzus persicae) contain mutations in NaCh. The pyrethroid-resistant kdr (794J) and super-kdr (UKO) strains contain mutations at residues L1014 and M918 in the channel α-subunit respectively, while the kdr + super-kdr strain (4824J), insensitive to pyrethroids, contains mutations at both L1014 and M918. Pl1a/GNA and Hv1a/GNA fusion proteins have estimated LC50 values of 0.35 and 0.19 mg mL(-1) when fed to wild-type M. persicae. For insecticide-resistant aphids, LC50 for the Pl1a/GNA fusion protein increased by 2-6-fold, correlating with pyrethroid resistance (wild type < kdr < super-kdr < kdr + super-kdr strains). In contrast, LC50 for the Hv1a/GNA fusion protein showed limited correlation with pyrethroid resistance. Mutations in the sodium channel in pyrethroid-resistant aphids also protect against a fusion protein containing a sodium-channel-specific toxin, in spite of differences in ligand-channel interactions, but do not confer resistance to a fusion protein targeting calcium channels. The use of fusion proteins with differing targets could play a role in managing pesticide resistance. © 2014 Society of Chemical Industry.

  16. Purification and subunit structure of a putative K sup + -channel protein identified by its binding properties for dendrotoxin I

    Energy Technology Data Exchange (ETDEWEB)

    Rehm, H.; Lazdunski, M. (Centre National de la Recherche Scientifique, Nice (France))

    1988-07-01

    The binding protein for the K{sup +}-channel toxin dendrotoxin I was purified from a detergent extract of rat brain membranes. The purification procedure utilized chromatography on DEAE-Trisacryl, affinity chromatography on a dendrotoxin-I-Aca 22 column, and wheat germ agglutinin-Affigel 10 with a final 3,800- to 4,600-fold enrichment and a recovery of 8-16%. The high affinity (K{sub d}, 40-100 pM) and specificity of the binding site are retained throughout the purification procedure. Analysis of the purified material on silver-stained NaDodSO{sub 4}/polyacrylamide gel revealed three bands of M{sub r} 76,000-80,000, 38,000 and 35,000. Interestingly, the binding site for {sup 125}I-labeled mast cell degranulating peptide, another putative K{sup +}-channel ligand from bee venom, which induces long-term potentiation in hippocampus, seems to reside on the same protein complex, as both binding sites copurify through the entire purification protocol.

  17. Purification and subunit structure of a putative K+-channel protein identified by its binding properties for dendrotoxin I

    International Nuclear Information System (INIS)

    Rehm, H.; Lazdunski, M.

    1988-01-01

    The binding protein for the K + -channel toxin dendrotoxin I was purified from a detergent extract of rat brain membranes. The purification procedure utilized chromatography on DEAE-Trisacryl, affinity chromatography on a dendrotoxin-I-Aca 22 column, and wheat germ agglutinin-Affigel 10 with a final 3,800- to 4,600-fold enrichment and a recovery of 8-16%. The high affinity (K d , 40-100 pM) and specificity of the binding site are retained throughout the purification procedure. Analysis of the purified material on silver-stained NaDodSO 4 /polyacrylamide gel revealed three bands of M r 76,000-80,000, 38,000 and 35,000. Interestingly, the binding site for 125 I-labeled mast cell degranulating peptide, another putative K + -channel ligand from bee venom, which induces long-term potentiation in hippocampus, seems to reside on the same protein complex, as both binding sites copurify through the entire purification protocol

  18. An updated and further developed theory and evidence for the close-contact, one-on-one association of nearly all cell K+ with beta- and gamma-carboxyl groups of intracellular proteins.

    Science.gov (United States)

    Ling, Gilbert N

    2005-01-01

    The primary focus of this communication is to present an updated and advanced version of the theory of close-contact association of molecules and ions through the spatial fixation and aggregation of the adsorbing sites. The last sections of the text also review a collection of relevant in vitro and in vivo experimental findings gathering since seventy years ago. Though some of these findings were published before the theory, old and new, they all support the theory that close-contact association with the beta-, and gamma-carboxyl groups of intracellular proteins causes the selective accumulation of potassium ions (K+) in living cells.

  19. Protein and cell patterning in closed polymer channels by photoimmobilizing proteins on photografted poly(ethylene glycol) diacrylate

    DEFF Research Database (Denmark)

    Larsen, Esben Kjær Unmack; Mikkelsen, Morten Bo Lindholm; Larsen, Niels Bent

    2014-01-01

    on uncoated polymer surfaces. The coating could also efficiently suppress the adhesion of mammalian cells as demonstrated using the HT-29 cancer cell line. In a subsequent equivalent process step, protein in aqueous solution could be anchored onto the PEGDA coating in spatially defined patterns...

  20. Role of the ERC motif in the proximal part of the second intracellular loop and the C-terminal domain of the human prostaglandin F2alpha receptor (hFP-R) in G-protein coupling control.

    Science.gov (United States)

    Pathe-Neuschäfer-Rube, Andrea; Neuschäfer-Rube, Frank; Püschel, Gerhard P

    2005-05-15

    The human FP-R (F2alpha prostaglandin receptor) is a Gq-coupled heptahelical ectoreceptor, which is of significant medical interest, since it is a potential target for the treatment of glaucoma and preterm labour. On agonist exposure, it mediates an increase in intracellular inositol phosphate formation. Little is known about the structures that govern the agonist-dependent receptor activation. In other prostanoid receptors, the C-terminal domain has been inferred in the control of agonist-dependent receptor activation. A DRY motif at the beginning of the second intracellular loop is highly conserved throughout the G-protein-coupled receptor family and appears to be crucial for controlling agonist-dependent receptor activation. It is replaced by an ERC motif in the FP-R and no evidence for the relevance of this motif in ligand-dependent activation of prostanoid receptors has been provided so far. The aim of the present study was to elucidate the potential role of the C-terminal domain and the ERC motif in agonist-controlled intracellular signalling in FP-R mutants generated by site-directed mutagenesis. It was found that substitution of the acidic Glu(132) in the ERC motif by a threonine residue led to full constitutive activation, whereas truncation of the receptor's C-terminal domain led to partial constitutive activation of all three intracellular signal pathways that had previously been shown to be activated by the FP-R, i.e. inositol trisphosphate formation, focal adhesion kinase activation and T-cell factor signalling. Inositol trisphosphate formation and focal adhesion kinase phosphorylation were further enhanced by ligand binding in cells expressing the truncation mutant but not the E132T (Glu132-->Thr) mutant. Thus C-terminal truncation appeared to result in a receptor with partial constitutive activation, whereas substitution of Glu132 by threonine apparently resulted in a receptor with full constitutive activity.

  1. Stochastic models of intracellular calcium signals

    Energy Technology Data Exchange (ETDEWEB)

    Rüdiger, Sten, E-mail: sten.ruediger@physik.hu-berlin.de

    2014-01-10

    Cellular signaling operates in a noisy environment shaped by low molecular concentrations and cellular heterogeneity. For calcium release through intracellular channels–one of the most important cellular signaling mechanisms–feedback by liberated calcium endows fluctuations with critical functions in signal generation and formation. In this review it is first described, under which general conditions the environment makes stochasticity relevant, and which conditions allow approximating or deterministic equations. This analysis provides a framework, in which one can deduce an efficient hybrid description combining stochastic and deterministic evolution laws. Within the hybrid approach, Markov chains model gating of channels, while the concentrations of calcium and calcium binding molecules (buffers) are described by reaction–diffusion equations. The article further focuses on the spatial representation of subcellular calcium domains related to intracellular calcium channels. It presents analysis for single channels and clusters of channels and reviews the effects of buffers on the calcium release. For clustered channels, we discuss the application and validity of coarse-graining as well as approaches based on continuous gating variables (Fokker–Planck and chemical Langevin equations). Comparison with recent experiments substantiates the stochastic and spatial approach, identifies minimal requirements for a realistic modeling, and facilitates an understanding of collective channel behavior. At the end of the review, implications of stochastic and local modeling for the generation and properties of cell-wide release and the integration of calcium dynamics into cellular signaling models are discussed.

  2. Natural channel protein inserts and functions in a completely artificial, solid-supported bilayer membrane

    OpenAIRE

    Zhang, Xiaoyan; Fu, Wangyang; Palivan, Cornelia G.; Meier, Wolfgang

    2013-01-01

    Reconstitution of membrane proteins in artificial membrane systems creates a platform for exploring their potential for pharmacological or biotechnological applications. Previously, we demonstrated amphiphilic block copolymers as promising building blocks for artificial membranes with long-term stability and tailorable structural parameters. However, the insertion of membrane proteins has not previously been realized in a large-area, stable, and solid-supported artificial membrane. Here, we s...

  3. Polyethylenimine modified poly(ethylene terephthalate) capillary channeled-polymer fibers for anion exchange chromatography of proteins.

    Science.gov (United States)

    Jiang, Liuwei; Jin, Yi; Marcus, R Kenneth

    2015-09-04

    Native poly(ethylene terephthalate) (PET) capillary-channeled polymer (C-CP) fibers have been previously studied as stationary phases for reversed phase and affinity protein separations. In this study, surface modified PET C-CP fibers were evaluated for the anion exchange separation of proteins. The native PET C-CP fibers were aminated using polyethylenimine (PEI) followed by a 1,4-butanediol diglycidyl ether (BUDGE) cross-linking step. Subsequent PEI/BUDGE treatments can be employed to further develop the polyamine layer on the fiber surfaces. The PEI densities of the modified fibers were quantified through the ninhydrin reaction, yielding values of 0.43-0.89μmolg(-1). The surface modification impact on column permeability was found to be 0.66×10(-11) to 1.33×10(-11)m(2), depending on the modification time and conditions. The dynamic binding capacities of the modified fiber media were determined to be 1.99-8.54mgmL(-1) bed volume, at linear velocities of 88-438cmmin(-1) using bovine serum albumin as the model protein. It was found that increasing the mobile phase linear velocity (up to 438cmmin(-1)) had no effect on the separation quality for a synthetic protein mixture, reflecting the lack of van Deemter C-term effects for the C-CP fiber phase. The low-cost, easy modification method and the capability of fast protein separation illustrate great potential in the use of PEI/BUDGE-modified PET C-CP fibers for high-throughput protein separation and downstream processing. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Effects of calcium channel on ovarian cancer cells.

    Science.gov (United States)

    Zhang, Chunyun; Li, Hailing

    2017-12-01

    The aim of the present study was to examine the effects of calcium channel protein on ovarian cancer cells. The expression of calcium channel protein in normal ovarian cells and ovarian cancer cells was detected by fluorescence quantitative PCR. Subsequently, the ovarian cancer cells were added to calcium channel protein activator media at various concentrations of 0, 1, 4, 8, 12, 16 and 20 mmol/l. The concentration of calcium ion in different samples was produced, and using an MTT assay, ovarian cancer cell activity in various samples was detected. Finally, a flow cytometer was used to explore the apoptosis rate. It was found that there was a significant difference between the expression of calcium channel protein in normal ovarian tissue and ovarian cancer cells (Pcancer cells to a certain extent, in a dose-dependent manner, especially when the concentration was at 12 mmol/l at which the intracellular calcium concentration was similar to that in normal ovarian cells. In conclusion, calcium ions play an important role in promoting cell proliferation of ovarian cancer cells, and they were involved in apoptosis of ovarian cancer cells to some extent, which regulates apoptosis by controlling the content of intracellular calcium.

  5. Yeast acyl-CoA-binding protein: acyl-CoA-binding affinity and effect on intracellular acyl-CoA pool size

    DEFF Research Database (Denmark)

    Knudsen, J; Faergeman, N J; Skøtt, H

    1994-01-01

    Acyl-CoA-binding protein (ACBP) is a 10 kDa protein characterized in vertebrates. We have isolated two ACBP homologues from the yeast Saccharomyces carlsbergensis, named yeast ACBP types 1 and 2. Both proteins contain 86 amino acid residues and are identical except for four conservative substitut...

  6. DMPD: Manipulation of mitogen-activated protein kinase/nuclear factor-kappaB-signalingcascades during intracellular Toxoplasma gondii infection. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15361242 Manipulation of mitogen-activated protein kinase/nuclear factor-kappaB-sig...mmunol Rev. 2004 Oct;201:191-205. (.png) (.svg) (.html) (.csml) Show Manipulation of mitogen-activated protein kinase/nuclear... gondii infection. PubmedID 15361242 Title Manipulation of mitogen-activated protein kinase/nuclear factor-k

  7. Membrane mechanisms and intracellular signalling in cell volume regulation

    DEFF Research Database (Denmark)

    Hoffmann, Else Kay; Dunham, Philip B.

    1995-01-01

    Volume regulation, Signal transduction, Calcium-calmodulin, Stretch-activated channels, Eicosanoids, Macromolecular crowding, Cytoskeleton, Protein phosphorylation, dephosphorylation.......Volume regulation, Signal transduction, Calcium-calmodulin, Stretch-activated channels, Eicosanoids, Macromolecular crowding, Cytoskeleton, Protein phosphorylation, dephosphorylation....

  8. Intracellular transport: from physics to ... biology.

    Science.gov (United States)

    Roux, Aurélien; Cuvelier, Damien; Bassereau, Patricia; Goud, Bruno

    2008-03-01

    Considerable effort over the past three decades has allowed the identification of the protein families that control the cellular machinery responsible for intracellular transport within eukaryotic cells. These proteins are estimated to represent about 10-20% of the human "proteome." The complexity of intracellular transport makes useful the development of model membranes. We describe here experimental systems based on lipid giant unilamellar vesicles (GUVs), which are attached to kinesin molecules. These systems give rise to thin membrane tubes and to complex tubular networks when incubated in vitro with microtubules and ATP. This type of assay, which mimics key events occurring during intracellular transport, allows physicists and biologists to understand how the unique mechanical properties of lipid membranes could be involved in the budding process, the sorting of cargo proteins and lipids, and the separation of the buds from a donor membrane.

  9. Spatial Cell Biology : Dissecting and directing intracellular transport mechanisms

    NARCIS (Netherlands)

    Adrian, M.

    2017-01-01

    Cellular compartmentalization and intracellular transport mechanisms are important to establish and maintain the spatial organisation of proteins and organelles needed to ensure proper cellular functioning. Especially in polarized cells like neurons, the proper distribution of proteins into the

  10. Stochastic models of intracellular transport

    KAUST Repository

    Bressloff, Paul C.

    2013-01-09

    The interior of a living cell is a crowded, heterogenuous, fluctuating environment. Hence, a major challenge in modeling intracellular transport is to analyze stochastic processes within complex environments. Broadly speaking, there are two basic mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually in the form of adenosine triphosphate hydrolysis, and can be direction specific, allowing biomolecules to be transported long distances; this is particularly important in neurons due to their complex geometry. In this review a wide range of analytical methods and models of intracellular transport is presented. In the case of diffusive transport, narrow escape problems, diffusion to a small target, confined and single-file diffusion, homogenization theory, and fractional diffusion are considered. In the case of active transport, Brownian ratchets, random walk models, exclusion processes, random intermittent search processes, quasi-steady-state reduction methods, and mean-field approximations are considered. Applications include receptor trafficking, axonal transport, membrane diffusion, nuclear transport, protein-DNA interactions, virus trafficking, and the self-organization of subcellular structures. © 2013 American Physical Society.

  11. Long-term fish oil supplementation induces cardiac electrical remodeling by changing channel protein expression in the rabbit model.

    Directory of Open Access Journals (Sweden)

    Xulin Xu

    2010-04-01

    Full Text Available Clinical trials and epidemiological studies have suggested that dietary fish oil (FO supplementation can provide an anti-arrhythmic benefit in some patient populations. The underlying mechanisms are not entirely clear. We wanted to understand how FO supplementation (for 4 weeks affected the action potential configuration/duration of ventricular myocytes, and the ionic mechanism(s/molecular basis for these effects. The experiments were conducted on adult rabbits, a widely used animal model for cardiac electrophysiology and pathophysiology. We used gas chromatography-mass spectroscopy to confirm that FO feeding produced a marked increase in the content of n-3 polyunsaturated fatty acids in the phospholipids of rabbit hearts. Left ventricular myocytes were used in current and voltage clamp experiments to monitor action potentials and ionic currents, respectively. Action potentials of myocytes from FO-fed rabbits exhibited much more positive plateau voltages and prolonged durations. These changes could be explained by an increase in the L-type Ca current (I(CaL and a decrease in the transient outward current (I(to in these myocytes. FO feeding did not change the delayed rectifier or inward rectifier current. Immunoblot experiments showed that the FO-feeding induced changes in I(CaL and I(to were associated with corresponding changes in the protein levels of major pore-forming subunits of these channels: increase in Cav1.2 and decrease in Kv4.2 and Kv1.4. There was no change in other channel subunits (Cav1.1, Kv4.3, KChIP2, and ERG1. We conclude that long-term fish oil supplementation can impact on cardiac electrical activity at least partially by changing channel subunit expression in cardiac myocytes.

  12. Potassium Channel Interacting Protein 2 (KChIP2) is not a transcriptional regulator of cardiac electrical remodeling

    DEFF Research Database (Denmark)

    Winther, Sine V; Tuomainen, Tomi; Borup, Rehannah

    2016-01-01

    The heart-failure relevant Potassium Channel Interacting Protein 2 (KChIP2) augments CaV1.2 and KV4.3. KChIP3 represses CaV1.2 transcription in cardiomyocytes via interaction with regulatory DNA elements. Hence, we tested nuclear presence of KChIP2 and if KChIP2 translocates into the nucleus...... in a Ca(2+) dependent manner. Cardiac biopsies from human heart-failure patients and healthy donor controls showed that nuclear KChIP2 abundance was significantly increased in heart failure; however, this was secondary to a large variation of total KChIP2 content. Administration of ouabain did...... not increase KChIP2 content in nuclear protein fractions in anesthetized mice. KChIP2 was expressed in cell lines, and Ca(2+) ionophores were applied in a concentration- and time-dependent manner. The cell lines had KChIP2-immunoreactive protein in the nucleus in the absence of treatments to modulate...

  13. Bipolar phospholipid sensing by TRPC5 calcium channel.

    Science.gov (United States)

    Beech, D J

    2007-02-01

    TRPC5 [TRP (transient receptor potential) canonical (or classical) 5] is a widely expressed mammalian homologue of Drosophila TRP, forming a calcium- and sodium-permeable channel in the plasma membrane either as a homomultimer or heteromultimer with other proteins (e.g. TRPC1). Although several factors are known to stimulate the channel, understanding of its endogenous activators and functions is limited. This paper provides a brief and focused review of our latest findings that show that TRPC5 is a sensor of important signalling phospholipids, including lysophosphatidylcholine and sphingosine 1-phosphate, acting extracellularly or intracellularly. Underlying mechanisms of action and biological relevance are discussed.

  14. Definitive localization of intracellular proteins: Novel approach using CRISPR-Cas9 genome editing, with glucose 6-phosphate dehydrogenase as a model.

    Science.gov (United States)

    Spencer, Netanya Y; Yan, Ziying; Cong, Le; Zhang, Yulong; Engelhardt, John F; Stanton, Robert C

    2016-02-01

    Studies to determine subcellular localization and translocation of proteins are important because subcellular localization of proteins affects every aspect of cellular function. Such studies frequently utilize mutagenesis to alter amino acid sequences hypothesized to constitute subcellular localization signals. These studies often utilize fluorescent protein tags to facilitate live cell imaging. These methods are excellent for studies of monomeric proteins, but for multimeric proteins, they are unable to rule out artifacts from native protein subunits already present in the cells. That is, native monomers might direct the localization of fluorescent proteins with their localization signals obliterated. We have developed a method for ruling out such artifacts, and we use glucose 6-phosphate dehydrogenase (G6PD) as a model to demonstrate the method's utility. Because G6PD is capable of homodimerization, we employed a novel approach to remove interference from native G6PD. We produced a G6PD knockout somatic (hepatic) cell line using CRISPR-Cas9 mediated genome engineering. Transfection of G6PD knockout cells with G6PD fluorescent mutant proteins demonstrated that the major subcellular localization sequences of G6PD are within the N-terminal portion of the protein. This approach sets a new gold standard for similar studies of subcellular localization signals in all homodimerization-capable proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Molecular cloning, characterization and analysis of the intracellular localization of a water-soluble chlorophyll-binding protein (WSCP) from Virginia pepperweed (Lepidium virginicum), a unique WSCP that preferentially binds chlorophyll b in vitro.

    Science.gov (United States)

    Takahashi, Shigekazu; Yanai, Haruna; Oka-Takayama, Yuko; Zanma-Sohtome, Aya; Fujiyama, Kosaku; Uchida, Akira; Nakayama, Katsumi; Satoh, Hiroyuki

    2013-12-01

    Various plants possess non-photosynthetic, hydrophilic chlorophyll (Chl) proteins called water-soluble Chl-binding proteins (WSCPs). WSCPs are categorized into two classes; Class I (photoconvertible type) and Class II (non-photoconvertible type). Among Class II WSCPs, only Lepidium virginicum WSCP (LvWSCP) exhibits a low Chl a/b ratio compared with that found in the leaf. Although the physicochemical properties of LvWSCP have been characterized, its molecular properties have not yet been documented. Here, we report the characteristics of the LvWSCP gene, the biochemical properties of a recombinant LvWSCP, and the intracellular localization of LvWSCP. The cloned LvWSCP gene possesses a 669-bp open reading frame. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis revealed that the precursor of LvWSCP contains both N- and C-terminal extension peptides. RT-PCR analysis revealed that LvWSCP was transcribed in various tissues, with the levels being higher in developing tissues. A recombinant LvWSCP and hexa-histidine fusion protein (LvWSCP-His) could