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Sample records for 91-kda membrane-bound protein

  1. Using supported bilayers to study the spatiotemporal organization of membrane bound proteins

    PubMed Central

    Field, Christine M.; Groen, Aaron C.; Mitchison, Timothy J.

    2015-01-01

    Cell division in prokaryotes and eukaryotes is commonly initiated by the well-controlled binding of proteins to the cytoplasmic side of the cell membrane. However, a precise characterization of the spatiotemporal dynamics of membrane-bound proteins is often difficult to achieve in vivo. Here, we present protocols for the use of supported lipid bilayers to rebuild the cytokinetic machineries of cells with greatly different dimensions: the bacterium Escherichia coli and eggs of the vertebrate Xenopus laevis. Combined with total internal reflection fluorescence (TIRF) microscopy, these experimental setups allow for precise quantitative analyses of membrane-bound proteins. The protocols described to obtain glass-supported membranes from bacterial and vertebrate lipids can be used as starting points for other reconstitution experiments. We believe that similar biochemical assays will be instrumental to study the biochemistry and biophysics underlying a variety of complex cellular tasks, such as signaling, vesicle trafficking and cell motility. PMID:25997350

  2. Non-denaturing gel electrophoresis system for the purification of membrane bound proteins

    SciTech Connect

    Cavinato, A.G.; Macleod, R.M.; Ahmed, M.S.

    1988-01-01

    A new method is described for the purification of a membrane bound glycoprotein, the kappa opioid receptor from human placental tissue. The method uses preparative slab-gel electrophoresis in the presence of the non-denaturing detergent CHAPS. A linear relationship between log molecular weight and SDS PAGE electrophoretic mobility of known molecular weight markers, in the presence of CHAPS, is observed. Using this method, we were able partially to purify an /sup 3/H-etorphine binding glycoprotein, from placental villus tissue, with an apparent molecular weight range of 60-70,000. The iodinated glycoprotein migrates in SDS PAGE with an apparent molecular weight of 63,000. This method may be useful for the isolation of membrane bound proteins, especially when an affinity ligand is not available.

  3. Next generation SPR technology of membrane-bound proteins for ligand screening and biomarker discovery

    PubMed Central

    Maynard, Jennifer A.; Lindquist, Nathan C.; Sutherland, Jamie N.; Lesuffleur, Antoine; Warrington, Arthur E.; Rodriguez, Moses; Oh, Sang-Hyun

    2009-01-01

    Technology based on surface plasmon resonance (SPR) has allowed rapid, label-free characterization of protein-protein and protein-small molecule interactions, from quantitative measurements of binding kinetics and thermodynamics and concentrations in complex samples to epitope analysis. SPR has become the gold standard in industrial and academic settings, in which typically the interaction between a pair of soluble binding partners is characterized in detail or a library of molecules is screened for binding against a single soluble protein. In spite of these successes, the technology is only beginning to be adapted to the needs of membrane-bound proteins. Including G protein-coupled receptors (GPCR), ion channels and other growth, immune and cellular receptors, these proteins are difficult to study in situ but represent promising targets for drug and biomarker development. Existing technologies, such as BIAcore™, have been adapted for membrane protein analysis by building supported lipid layers or vesicle capture on existing chips. Newer technologies, still in development, will allow membrane proteins to be presented in native or near-native formats. These include SPR nanopore arrays, in which lipid bilayers containing membrane proteins stably span small pores that are addressable from both sides of the bilayer. Here, we discuss successes with current SPR instrumentation and the potential for SPR nanopore arrays to enable quantitative, high-throughput screening of GPCR ligands, biomarker discovery involving membrane bound proteins and basic cellular biology. PMID:19918786

  4. Biochemical similarities between soluble and membrane-bound calcium-dependent protein kinases of barley

    SciTech Connect

    Klimczak, L.J.; Hind, G. )

    1990-04-01

    The soluble and membrane-bound forms of the calcium-dependent protein kinase from barley leaves (Hordeum vulgare L. cv. Borsoy) have been partially purified and compared. Both forms showed an active polypeptide of 37 kilodaltons on activity gels with incorporated histone as substrate. They eluted from chromatofocusing columns at an identical isoelectric point of pH 4.25 {plus minus} 0.2, and also comigrated on several other chromatographic affinity media including Matrex Gel Blue A, histone-agarose, phenyl-Sepharose, and heparin-agarose. Both activities comigrated with chicken ovalbumin during gel filtration through Sephacryl S-200, indicating a native molecular mass of 45 kilodaltons. The activities share a number of enzymatic properties including salt and pH dependence, free calcium stimulation profile, substrate specificity, and Km values. The soluble activity was shown to bind to artificial lipid vesicles. These data suggest strongly that the soluble and membrane-bound calcium-dependent protein kinases from barley are very closely related or even identical.

  5. In Vitro Synthesis of Proteins by Membrane-Bound Polyribosomes from Vesicular Stomatitis Virus-Infected HeLa Cells

    PubMed Central

    Grubman, Marvin J.; Ehrenfeld, Ellie; Summers, Donald F.

    1974-01-01

    Membrane-bound polysomes from vesicular stomatitis virus (VSV)-infected HeLa cells synthesize predominantly three proteins in an in vitro protein synthesizing system. These three proteins have different molecular weights than the viral structural proteins, i.e., 115,000, 88,000, and 72,000. Addition of preincubated L or HeLa cell S10 or HeLa cell crude initiation factors stimulates amino acid incorporation and, furthermore, alters the pattern of proteins synthesized. Stimulated membrane-bound polysomes synthesize predominantly viral protein G and lesser amounts of N, NS, and M. In vitro synthesized proteins G and N are very similar to virion proteins G and N based on analysis of tryptic methionine-labeled peptides. Most methionine-labeled tryptic peptides of virion G protein contain no carbohydrate moieties, since about 90% of sugar-labeled peptides co-chromatograph with only about 10% of methionine-labeled peptides. Sucrose gradient analysis of the labeled RNA present in VSV-infected membrane-bound polysomes reveals a relative enrichment in a class of viral RNA sedimenting slightly faster than the total population of the 13 to 15S mRNA, as compared to a VSV-infected crude cytoplasmic extract. A number of proteins, other than the viral structural proteins, are synthesized in the cytoplasm of five lines of VSV-infected cells. One of these proteins has the same molecular weight as the major in vitro synthesized protein, P88. In vitro synthesized protein P88 does not appear to be a precursor of viral structural proteins G, N, or M based on pulse-chase experiments and tryptic peptide mapping. Nonstimulated membrane-bound polysomes from uninfected HeLa cells synthesize the same size distribution of proteins as nonstimulated VSV-infected membrane-bound polysomes. Images PMID:4368799

  6. High-Yield Expression of a Catalytically Active Membrane-Bound Protein: Human P450 Oxidoreductase

    PubMed Central

    Sandee, Duanpen

    2011-01-01

    P450 oxidoreductase (POR) is a two-flavin protein that reduces microsomal P450 enzymes and some other proteins. Preparation of active bacterially expressed human POR for biochemical studies has been difficult because membrane-bound proteins tend to interact with column matrices. To reduce column-protein interactions and permit more vigorous washing, human POR lacking 27 N-terminal residues (N-27 POR) was modified to carry a C-terminal Gly3His6-tag (N-27 POR-G3H6). When expressed in Escherichia coli, N-27 POR-G3H6 could be purified to apparent homogeneity by a modified, single-step nickel-nitrilotriacetic acid affinity chromatography, yielding 31 mg POR per liter of culture, whereas standard purification of native N-27 POR required multiple steps, yielding 5 mg POR per liter. Both POR proteins had absorption maxima at 375 and 453 nm and both reduced cytochrome c with indistinguishable specific activities. Using progesterone as substrate for bacterially expressed purified human P450c17, the Michaelis constant for 17α-hydroxylase activity supported by N-27 POR or N-27 POR-G3H6 were 1.73 or 1.49 μm, and the maximal velocity was 0.029 or 0.026 pmol steroids per picomole P450 per minute, respectively. Using 17-hydroxypregnenolone as the P450c17 substrate, the Michaelis constant for 17,20 lyase activity using N-27 POR or N-27 POR-G3H6 was 1.92 or 1.89 μm and the maximal velocity was 0.041 or 0.042 pmol steroid per picomole P450 per minute, respectively. Thus, N-27 POR-G3H6 is equally active as native N-27 POR. This expression and purification system permits the rapid preparation of large amounts of highly pure, biologically active POR and may be generally applicable for the preparation of membrane-bound proteins. PMID:21586563

  7. Membrane-bound tomato mosaic virus replication proteins participate in RNA synthesis and are associated with host proteins in a pattern distinct from those that are not membrane bound.

    PubMed

    Nishikiori, Masaki; Dohi, Koji; Mori, Masashi; Meshi, Tetsuo; Naito, Satoshi; Ishikawa, Masayuki

    2006-09-01

    Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNA-dependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positive-strand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130- and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the non-membrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidylcholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity. PMID:16912296

  8. Exploring homo-FRET to quantify the oligomer stoichiometry of membrane-bound proteins involved in a cooperative partition equilibrium.

    PubMed

    Melo, Ana M; Fedorov, Aleksander; Prieto, Manuel; Coutinho, Ana

    2014-09-14

    The establishment of protein-protein interactions between membrane-bound proteins is associated with several biological functions and dysfunctions. Here, an analytical framework that uses energy homo transfer to directly probe quantitatively the oligomerization state of membrane-bound proteins engaged in a three-state cooperative partition is presented. Briefly, this model assumes that monomeric protein molecules partition into the bilayer surface and reversibly assemble into oligomers with k subunits. A general equation relating the overall steady-state fluorescence anisotropy of the sample to its fractional labeling was derived by considering explicitly that the anisotropy of mixed oligomers containing i-labeled monomers is inversely proportional to the number of labeled subunits per oligomer (Runnels and Scarlata limit). This method was very robust in describing the electrostatic interaction of Alexa Fluor 488 fluorescently labeled lysozyme (Lz-A488) with phosphatidylserine-containing membranes. The pronounced decrease detected in the fluorescence anisotropy of Lz-A488 always correlated with the system reaching a high membrane surface density of the protein (at a low lipid-to-protein (L/P) molar ratio). The occurrence of energy homo transfer-induced fluorescence depolarization was further confirmed by measuring the anisotropy decays of Lz-A488 under these conditions. A global analysis of the steady-state anisotropy data obtained under a wide range of experimental conditions (variable anionic lipid content of the liposomes, L/P molar ratios and protein fractional labeling) confirmed that membrane-bound Lz-A488 assembled into oligomeric complexes, possibly with a stoichiometry of k = 6 ± 1. This study illustrates that even in the presence of a coupled partition-oligomerization equilibrium, steady-state anisotropy measurements provide a simple and reliable tool to monitor the self-assembly of membrane-bound proteins. PMID:24722583

  9. Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

    PubMed

    Zhu, Haiyan; Fang, Xiaoyun; Zhang, Dongmei; Wu, Weicheng; Shao, Miaomiao; Wang, Lan; Gu, Jianxin

    2016-01-01

    Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system. PMID:26481477

  10. Identification and characterization of novel membrane-bound PRL protein tyrosine phosphatases from Setaria cervi, a bovine filarial parasite.

    PubMed

    Singh, Neetu; Yadav, Smita; Rathaur, Sushma

    2015-11-01

    A significant amount of protein tyrosine phosphatase (PTP) activity was detected in the detergent-soluble membrane-bound fraction of Setaria cervi, a bovine filarial parasite. The membrane-bound PTP activity was significantly inhibited when the adult parasites were exposed to compounds having antifilarial activity like aspirin and SK7 as well as phenylarsine oxide, a specific PTP inhibitor suggesting that this activity is stress regulated. Further, this enzyme was purified as a single protein of apparently 21 kDa using two different chromatographic techniques. The MALDI-MS/MS analysis of its peptides showed closest match with protein tyrosine phosphatase PRL (Aedes aegypti). This purified enzyme (named as PRL) showed maximum activity at pH 5.5/37 °C and hydrolysed para nitro phenyl phosphate (pNPP) at the highest rate followed by O-P-L-tyrosine and O-P-L-threonine. It showed significant inhibition by specific inhibitors of PTP such as sodium orthovanadate, phenylarsine oxide and ammonium molybdate and was activated by dithiothreitol (DTT). The active site modification studies suggested involvement of cysteine, arginine, histidine and aspartic acid in the catalytic activity of PRL. The activity of S. cervi PRL was also found to be resistant towards the external oxidative stress. Thus, S. cervi PRL could be taken as a potential target for the management of human lymphatic filariasis. PMID:26341797

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

    PubMed Central

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

    1995-01-01

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

  12. Nanoscale patterning of membrane-bound proteins formed through curvature-induced partitioning of phase-specific receptor lipids.

    PubMed

    Ogunyankin, Maria O; Huber, Dale L; Sasaki, Darryl Y; Longo, Marjorie L

    2013-05-21

    This work describes a technique for forming high-density arrays and patterns of membrane-bound proteins through binding to a curvature-organized compositional pattern of metal-chelating lipids (Cu(2+)-DOIDA or Cu(2+)-DSIDA). In this bottom-up approach, the underlying support is an e-beam formed, square lattice pattern of hemispheres. This curvature pattern sorts Cu(2+)-DOIDA to the 200 nm hemispherical lattice sites of a 600 nm × 600 nm unit cell in Ld - Lo phase separated lipid multibilayers. Binding of histidine-tagged green fluorescent protein (His-GFP) creates a high density array of His-GFP-bound pixels localized to the square lattice sites. In comparison, the negative pixel pattern is created by sorting Cu(2+)-DSIDA in Ld - Lβ' phase separated lipid multibilayers to the flat grid between the lattice sites followed by binding to His-GFP. Lattice defects in the His-GFP pattern lead to interesting features such as pattern circularity. We also observe defect-free arrays of His-GFP that demonstrate perfect arrays can be formed by this method suggesting the possibility of using this approach for the localization of various active molecules to form protein, DNA, or optically active molecular arrays. PMID:23642033

  13. Membrane-bound states of alpha-lactalbumin: implications for the protein stability and conformation.

    PubMed Central

    Cawthern, K. M.; Permyakov, E.; Berliner, L. J.

    1996-01-01

    alpha-Lactalbumin (alpha-LA) associates with dimyristoylphosphatidylcholine (DMPC) or egg lecithin (EPC) liposomes. Thermal denaturation of isolated DMPC or EPC alpha-LA complexes was dependent on the metal bound state of the protein. The intrinsic fluorescence of thermally denatured DMPC-alpha-LA was sensitive to two thermal transitions: the Tc of the lipid vesicles, and the denaturation of the protein. Quenching experiments suggested that tryptophan accessibility increased upon protein-DMPC association, in contrast with earlier suggestions that the limited emission red shift upon association with the liposome was due to partial insertion of tryptophan into the apolar phase of the bilayer (Hanssens I et al., 1985, Biochim Biophys Acta 817:154-166). On the other hand, above the protein transition (70 degrees C), the spectral blue shifts and reduced accessibility to quencher suggested that tryptophan interacts significantly with the apolar phase of either DMPC and EPC. At pH 2, where the protein inserts into the bilayer rapidly, the isolated DMPC-alpha-LA complex showed a distinct fluorescence thermal transition between 40 and 60 degrees C, consistent with a partially inserted form that possesses some degree of tertiary structure and unfolds cooperatively. This result is significant in light of earlier findings of increased helicity for the acid form, i.e., molten globule state of the protein (Hanssens I et al., 1985, Biochim Biophys Acta 817:154-166). These results suggest a model where a limited expansion of conformation occurs upon association with the membrane at neutral pH and physiological temperatures, with a concomitant increase in the exposure of tryptophan to external quenchers; i.e., the current data do not support a model where an apolar, tryptophan-containing surface is covered by the lipid phase of the bilayer. PMID:8819172

  14. Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins

    PubMed Central

    Nijtmans, Leo G.J.; de Jong, Liesbeth; Artal Sanz, Marta; Coates, Philip J.; Berden, Jan A.; Willem Back, Jaap; Muijsers, Anton O.; van der Spek, Hans; Grivell, Les A.

    2000-01-01

    Prohibitins are ubiquitous, abundant and evolutionarily strongly conserved proteins that play a role in important cellular processes. Using blue native electrophoresis we have demonstrated that human prohibitin and Bap37 together form a large complex in the mitochondrial inner membrane. This complex is similar in size to the yeast complex formed by the homologues Phb1p and Phb2p. In yeast, levels of this complex are increased on co-overexpression of both Phb1p and Phb2p, suggesting that these two proteins are the only components of the complex. Pulse–chase experiments with mitochondria isolated from phb1/phb2-null and PHB1/2 overexpressing cells show that the Phb1/2 complex is able to stabilize newly synthesized mitochondrial translation products. This stabilization probably occurs through a direct interaction because association of mitochondrial translation products with the Phb1/2 complex could be demonstrated. The fact that Phb1/2 is a large multimeric complex, which provides protection of native peptides against proteolysis, suggests a functional homology with protein chaperones with respect to their ability to hold and prevent misfolding of newly synthesized proteins. PMID:10835343

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

    PubMed Central

    Gorfe, Alemayehu A.

    2015-01-01

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

  16. Studies of the molecular effects of a solid support upon lipid membranes and membrane bound proteins

    NASA Astrophysics Data System (ADS)

    Hartshorn, Christopher M.

    Often, membrane/protein systems are studied and/or utilized on solid supports. The underlying substrate in solid supported lipid bilayer assemblies causes large perturbations to the membrane, but the nature of these effects are not well understood. To gain an understanding, these effects were studied on two fronts: the effect upon the membrane by itself, and then the effects upon a membrane/protein system. First, all-atom molecular dynamics (MD) simulations of DLPC, DMPC, POPC, and DEPC on a hydroxylated nanocrystalline alpha-quartz (011) slab revealed a pronounced thinning effect in the lipid bilayers. It was shown that this thinning effect proceeded by one of two mechanisms: the first through a curling of the terminal methyl groups at the interface of the opposing leaflets, and the second through increased interdigitation of the alkyl chains. Also, with the introduction of the solid support, marked asymmetries in a number of structural properties were reported. These asymmetries included (a) the surface area per lipid, (b) the electron densities of the polar head groups, (c) the radial distributions of the choline groups, and (d) the average orientation of water surrounding the membranes. Next, the free energy perturbation method was used to begin calculating the change in free energy (DeltaGbinding) from a Gramicidin monomer to its dimeric state, which were simulated via MD of supported DLPC, DMPC, and DEPC bilayers. The most notable effect was an asymmetry of the calculated free energies relative to the bilayer side closest to the solid support. In all three systems, there was a large difference in free energy between the Gramicidin monomers that were close to the support and the monomers further from the support.

  17. The poliovirus receptor protein is produced both as membrane-bound and secreted forms.

    PubMed Central

    Koike, S; Horie, H; Ise, I; Okitsu, A; Yoshida, M; Iizuka, N; Takeuchi, K; Takegami, T; Nomoto, A

    1990-01-01

    Both genomic and complementary DNA clones encoding poliovirus receptors were isolated from genomic and complementary DNA libraries prepared from HeLa S3 cells, respectively. Nucleotide sequence analysis of these cloned DNAs revealed that the poliovirus receptor gene is approximately 20 kb long and contains seven introns in the coding region, and that at least four mRNA isoforms referring to the coding sequence are generated by alternative splicing and appear to encode four different molecules, that is, PVR alpha, PVR beta, PVR gamma and PVR delta. The predicted amino acid sequences indicate that PVR alpha and PVR delta, corresponding to the previously described cDNA clones H20A and H20B, respectively, are integral membrane proteins while the other two molecules described here for the first time lack a putative transmembrane domain. Mouse cell transformants carrying PVR alpha were permissive for poliovirus infection, but those carrying PVR beta were hardly permissive. In contrast to PVR alpha, PVR beta was not detected on the surface of the mouse cell transformants but was detected in the culture fluid by an immunological method using a monoclonal antibody against poliovirus receptor. Three types of splicing products for PVR alpha, PVR beta and PVR gamma were detected by polymerase chain reactions using appropriate primers in poly(A)+ RNAs of the brain, leukocyte, liver, lung and placenta of humans; the choice of primers used did not permit detection of PVR delta. In situ hybridization using a cDNA fragment as a probe demonstrated that the PVR gene is located at the band q13.1----13.2 of human chromosome 19. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2170108

  18. Structure and Dynamics of the Membrane-Bound Form of Pf1 Coat Protein: Implications of Structural Rearrangement for Virus Assembly

    PubMed Central

    Park, Sang Ho; Marassi, Francesca M.; Black, David; Opella, Stanley J.

    2010-01-01

    The three-dimensional structure of the membrane-bound form of the major coat protein of Pf1 bacteriophage was determined in phospholipid bilayers using orientation restraints derived from both solid-state and solution NMR experiments. In contrast to previous structures determined solely in detergent micelles, the structure in bilayers contains information about the spatial arrangement of the protein within the membrane, and thus provides insights to the bacteriophage assembly process from membrane-inserted to bacteriophage-associated protein. Comparisons between the membrane-bound form of the coat protein and the previously determined structural form found in filamentous bacteriophage particles demonstrate that it undergoes a significant structural rearrangement during the membrane-mediated virus assembly process. The rotation of the transmembrane helix (Q16–A46) around its long axis changes dramatically (by 160°) to obtain the proper alignment for packing in the virus particles. Furthermore, the N-terminal amphipathic helix (V2–G17) tilts away from the membrane surface and becomes parallel with the transmembrane helix to form one nearly continuous long helix. The spectra obtained in glass-aligned planar lipid bilayers, magnetically aligned lipid bilayers (bicelles), and isotropic lipid bicelles reflect the effects of backbone motions and enable the backbone dynamics of the N-terminal helix to be characterized. Only resonances from the mobile N-terminal helix and the C-terminus (A46) are observed in the solution NMR spectra of the protein in isotropic q > 1 bicelles, whereas only resonances from the immobile transmembrane helix are observed in the solid-state 1H/15N-separated local field spectra in magnetically aligned bicelles. The N-terminal helix and the hinge that connects it to the transmembrane helix are significantly more dynamic than the rest of the protein, thus facilitating structural rearrangement during bacteriophage assembly. PMID:20816058

  19. Incorporation of membrane-bound, mammalian-derived immunomodulatory proteins into influenza whole virus vaccines boosts immunogenicity and protection against lethal challenge

    PubMed Central

    Herbert, Andrew S; Heffron, Lynn; Sundick, Roy; Roberts, Paul C

    2009-01-01

    Background Influenza epidemics continue to cause morbidity and mortality within the human population despite widespread vaccination efforts. This, along with the ominous threat of an avian influenza pandemic (H5N1), demonstrates the need for a much improved, more sophisticated influenza vaccine. We have developed an in vitro model system for producing a membrane-bound Cytokine-bearing Influenza Vaccine (CYT-IVAC). Numerous cytokines are involved in directing both innate and adaptive immunity and it is our goal to utilize the properties of individual cytokines and other immunomodulatory proteins to create a more immunogenic vaccine. Results We have evaluated the immunogenicity of inactivated cytokine-bearing influenza vaccines using a mouse model of lethal influenza virus challenge. CYT-IVACs were produced by stably transfecting MDCK cell lines with mouse-derived cytokines (GM-CSF, IL-2 and IL-4) fused to the membrane-anchoring domain of the viral hemagglutinin. Influenza virus replication in these cell lines resulted in the uptake of the bioactive membrane-bound cytokines during virus budding and release. In vivo efficacy studies revealed that a single low dose of IL-2 or IL-4-bearing CYT-IVAC is superior at providing protection against lethal influenza challenge in a mouse model and provides a more balanced Th1/Th2 humoral immune response, similar to live virus infections. Conclusion We have validated the protective efficacy of CYT-IVACs in a mammalian model of influenza virus infection. This technology has broad applications in current influenza virus vaccine development and may prove particularly useful in boosting immune responses in the elderly, where current vaccines are minimally effective. PMID:19393093

  20. In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish.

    PubMed

    Lei, Li; Egli, Martin

    2016-01-01

    Fish and human cytochrome P450 (P450) 17A1 catalyze both steroid 17α-hydroxylation and 17α,20-lyase reactions. Fish P450 17A2 catalyzes only 17α-hydroxylation. Both enzymes are microsomal-type P450s, integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment, the signal anchor sequence. The presence of this N-terminal region renders expression of full-length proteins challenging or impossible. For some proteins, variable truncation of the signal anchor sequence precludes expression or results in poor expression levels. To crystallize P450 17A1 and 17A2 in order to gain insight into their different activities, we used an alternative N-terminal sequence to boost expression together with in situ proteolysis. Key features of our approach to identify crystallizable P450 fragments were the use of an N-terminal leader sequence, a screen composed of 12 proteases to establish optimal cleavage, variations of protease concentration in combination with an SDS-PAGE assay, and analysis of the resulting fragments using Edman sequencing. Described in this unit are protocols for vector preparation, expression, purification, and in situ proteolytic crystallization of two membrane-bound P450 proteins. © 2016 by John Wiley & Sons, Inc. PMID:27038268

  1. Complement S-protein (vitronectin) is associated with cytolytic membrane-bound C5b-9 complexes.

    PubMed Central

    Bhakdi, S; Käflein, R; Halstensen, T S; Hugo, F; Preissner, K T; Mollnes, T E

    1988-01-01

    It has been assumed that S-protein (vitronectin) associates with terminal C5b-9 complement complexes only when the latter fail to attach to target lipid bilayers, thereby forming inactive fluid-phase SC5b-9 complexes. Using monoclonal anti-S-protein antibodies, we show here that a minor portion of C5b-9 complexes associated with both homologous and heterologous cells contain S-protein. This conclusion derives from Western blot analyses, from the sedimentation behaviour of solubilized S-protein, and from the fact that the protein co-immunoprecipitates with C5b-9(m). Association of S-protein with C5b-9(m) takes place primarily at the stage of C9-binding. An average of less than or equal to 0.4 moles of S-protein are estimated to be present per mole C5b-9(m). Hence, only a fraction of C5b-9 complexes contain S-protein. The function of cell-bound S-protein is unknown. Haemolytic titrations with purified components failed to demonstrate any protective effect of S-protein on the lysis of sheep or human erythrocytes by C5b-9. S-protein bound to complement-lysed homologous or heterologous cells is readily detectable by conventional immunocytochemical staining. We conclude that differentiation between tissue-deposited fluid-phase C5b-9 and membrane C5b-9 complexes cannot be made on the basis of immunohistological stainings for S-protein alone. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:2466593

  2. Functional cooperation and separation of translocators in protein import into mitochondria, the double-membrane bounded organelles.

    PubMed

    Endo, Toshiya; Yamamoto, Hayashi; Esaki, Masatoshi

    2003-08-15

    Nearly all mitochondrial proteins are synthesized in the cytosol and subsequently imported into mitochondria with the aid of translocators: the TOM complex in the outer membrane, and the TIM23 and TIM22 complexes in the inner membrane. The TOM complex and the TIM complexes cooperate to achieve efficient transport of proteins to the matrix or into the inner membrane and several components, including Tom22, Tim23, Tim50 and small Tim proteins, mediate functional coupling of the two translocator systems. The TOM complex can be disconnected from the TIM systems and their energy sources (ATP and DeltaPsi), however, using alternative mechanisms to achieve vectorial protein translocation across the outer membrane PMID:12857785

  3. Conformation of membrane-bound proteins revealed by vacuum-ultraviolet circular-dichroism and linear-dichroism spectroscopy.

    PubMed

    Matsuo, Koichi; Maki, Yasuyuki; Namatame, Hirofumi; Taniguchi, Masaki; Gekko, Kunihiko

    2016-03-01

    Knowledge of the conformations of a water-soluble protein bound to a membrane is important for understanding the membrane-interaction mechanisms and the membrane-mediated functions of the protein. In this study we applied vacuum-ultraviolet circular-dichroism (VUVCD) and linear-dichroism (LD) spectroscopy to analyze the conformations of α-lactalbumin (LA), thioredoxin (Trx), and β-lactoglobulin (LG) bound to phosphatidylglycerol liposomes. The VUVCD analysis coupled with a neural-network analysis showed that these three proteins have characteristic helix-rich conformations involving several helical segments, of which two amphiphilic or hydrophobic segments take part in interactions with the liposome. The LD analysis predicted the average orientations of these helix segments on the liposome: two amphiphilic helices parallel to the liposome surface for LA, two hydrophobic helices perpendicular to the liposome surface for Trx, and a hydrophobic helix perpendicular to and an amphiphilic helix parallel to the liposome surface for LG. This sequence-level information about the secondary structures and orientations was used to formulate interaction models of the three proteins at the membrane surface. This study demonstrates the validity of a combination of VUVCD and LD spectroscopy in conformational analyses of membrane-binding proteins, which are difficult targets for X-ray crystallography and nuclear magnetic resonance spectroscopy. PMID:26756612

  4. Biosynthesis of the carbohydrate moieties of arabinogalactan proteins by membrane-bound β-glucuronosyltransferases from radish primary roots.

    PubMed

    Endo, Maya; Kotake, Toshihisa; Watanabe, Yoko; Kimura, Kazumasa; Tsumuraya, Yoichi

    2013-12-01

    A membrane fraction from etiolated 6-day-old primary radish roots (Raphanus sativus L. var hortensis) contained β-glucuronosyltransferases (GlcATs) involved in the synthesis of the carbohydrate moieties of arabinogalactan proteins (AGPs). The GlcATs transferred [(14)C]GlcA from UDP-[(14)C]GlcA on to β-(1 → 3)-galactan as an exogenous acceptor substrate, giving a specific activity of 50-150 pmol min(-1) (mg protein)(-1). The enzyme specimen also catalyzed the transfer of [(14)C]GlcA on to an enzymatically modified AGP from mature radish root. Analysis of the transfer products revealed that the transfer of [(14)C]GlcA occurred preferentially on to consecutive (1 → 3)-linked β-Gal chains as well as single branched β-(1 → 6)-Gal residues through β-(1 → 6) linkages, producing branched acidic side chains. The enzymes also transferred [(14)C]GlcA residues on to several oligosaccharides, such as β-(1 → 6)- and β-(1 → 3)-galactotrioses. A trisaccharide, α-L-Araf-(1 → 3)-β-Gal-(1 → 6)-Gal, was a good acceptor, yielding a branched tetrasaccharide, α-L-Araf-(1 → 3)[β-GlcA-(1 → 6)]-β-Gal-(1 → 6)-Gal. We report the first in vitro assay system for β-GlcATs involved in the AG synthesis as a step toward full characterization and cloning. PMID:24057431

  5. Inhibition of NADPH oxidase 1 activity and blocking the binding of cytosolic and membrane-bound proteins by honokiol inhibit migratory potential of melanoma cells.

    PubMed

    Prasad, Ram; Kappes, John C; Katiyar, Santosh K

    2016-02-16

    Overexpression of NADPH oxidase 1 (Nox1) in melanoma cells is often associated with increased migration/metastasis rate. To develop effective treatment options, we have examined the effect of honokiol, a phytochemical from Magnolia plant, on the migratory potential of human melanoma cell lines (A375, Hs294t, SK-Mel119 and SK-Mel28) and assessed whether Nox1 is the target. Using an in vitro cell migration assay, we observed that treatment of different melanoma cell lines with honokiol for 24 h resulted in a dose-dependent inhibition of cell migration that was associated with reduction in Nox1 expression and reduced levels of oxidative stress. Treatment of cells with N-acetyl-L-cysteine, an anti-oxidant, also inhibited the migration of melanoma cells. Treatment of cells with diphenyleneiodonium chloride, an inhibitor of Nox1, significantly decreased the migration ability of Hs294t and SK-Mel28 cells. Further, we examined the effect of honokiol on the levels of core proteins (p22phox and p47phox) of the NADPH oxidase complex. Treatment of Hs294t and SK-Mel28 cells with honokiol resulted in accumulation of the cytosolic p47phox protein and decreased levels of the membrane-bound p22phox protein, thus blocking their interaction and inhibiting Nox1 activation. Our in vivo bioluminescence imaging data indicate that oral administration of honokiol inhibited the migration/extravasation and growth of intravenously injected melanoma cells in internal body organs, such as liver, lung and kidney in nude mice, and that this was associated with an inhibitory effect on Nox1 activity in these internal organs/tissues. PMID:26760964

  6. Inhibition of NADPH oxidase 1 activity and blocking the binding of cytosolic and membrane-bound proteins by honokiol inhibit migratory potential of melanoma cells

    PubMed Central

    Prasad, Ram; Kappes, John C.; Katiyar, Santosh K.

    2016-01-01

    Overexpression of NADPH oxidase 1 (Nox1) in melanoma cells is often associated with increased migration/metastasis rate. To develop effective treatment options, we have examined the effect of honokiol, a phytochemical from Magnolia plant, on the migratory potential of human melanoma cell lines (A375, Hs294t, SK-Mel119 and SK-Mel28) and assessed whether Nox1 is the target. Using an in vitro cell migration assay, we observed that treatment of different melanoma cell lines with honokiol for 24 h resulted in a dose-dependent inhibition of cell migration that was associated with reduction in Nox1 expression and reduced levels of oxidative stress. Treatment of cells with N-acetyl-L-cysteine, an anti-oxidant, also inhibited the migration of melanoma cells. Treatment of cells with diphenyleneiodonium chloride, an inhibitor of Nox1, significantly decreased the migration ability of Hs294t and SK-Mel28 cells. Further, we examined the effect of honokiol on the levels of core proteins (p22phox and p47phox) of the NADPH oxidase complex. Treatment of Hs294t and SK-Mel28 cells with honokiol resulted in accumulation of the cytosolic p47phox protein and decreased levels of the membrane-bound p22phox protein, thus blocking their interaction and inhibiting Nox1 activation. Our in vivo bioluminescence imaging data indicate that oral administration of honokiol inhibited the migration/extravasation and growth of intravenously injected melanoma cells in internal body organs, such as liver, lung and kidney in nude mice, and that this was associated with an inhibitory effect on Nox1 activity in these internal organs/tissues. PMID:26760964

  7. The MUC4 membrane-bound mucin regulates esophageal cancer cell proliferation and migration properties: Implication for S100A4 protein

    SciTech Connect

    Bruyere, Emilie; Jonckheere, Nicolas; Frenois, Frederic; Mariette, Christophe; Van Seuningen, Isabelle

    2011-09-23

    Highlights: {yields} Loss of MUC4 reduces proliferation of esophageal cancer cells. {yields} MUC4 inhibition impairs migration of esophageal cancer cells but not their invasion. {yields} Loss of MUC4 significantly reduces in vivo tumor growth. {yields} Decrease of S100A4 induced by MUC4 inhibition impairs proliferation and migration. -- Abstract: MUC4 is a membrane-bound mucin known to participate in tumor progression. It has been shown that MUC4 pattern of expression is modified during esophageal carcinogenesis, with a progressive increase from metaplastic lesions to adenocarcinoma. The principal cause of development of esophageal adenocarcinoma is the gastro-esophageal reflux, and MUC4 was previously shown to be upregulated by several bile acids present in reflux. In this report, our aim was thus to determine whether MUC4 plays a role in biological properties of human esophageal cancer cells. For that stable MUC4-deficient cancer cell lines (shMUC4 cells) were established using a shRNA approach. In vitro (proliferation, migration and invasion) and in vivo (tumor growth following subcutaneous xenografts in SCID mice) biological properties of shMUC4 cells were analyzed. Our results show that shMUC4 cells were less proliferative, had decreased migration properties and did not express S100A4 protein when compared with MUC4 expressing cells. Absence of MUC4 did not impair shMUC4 invasiveness. Subcutaneous xenografts showed a significant decrease in tumor size when cells did not express MUC4. Altogether, these data indicate that MUC4 plays a key role in proliferative and migrating properties of esophageal cancer cells as well as is a tumor growth promoter. MUC4 mucin appears thus as a good therapeutic target to slow-down esophageal tumor progression.

  8. Domain Organization of Membrane-Bound Factor VIII

    PubMed Central

    Stoilova-McPhie, Svetla; Lynch, Gillian C.; Ludtke, Steven; Pettitt, B. Montgomery

    2014-01-01

    Factor VIII (FVIII) is the blood coagulation protein which when defective or deficient causes for hemophilia A, a severe hereditary bleeding disorder. Activated FVIII (FVIIIa) is the cofactor to the serine protease factor IXa (FIXa) within the membrane-bound Tenase complex, responsible for amplifying its proteolytic activity more than 100,000 times, necessary for normal clot formation. FVIII is composed of two noncovalently linked peptide chains: a light chain (LC) holding the membrane interaction sites and a heavy chain (HC) holding the main FIXa interaction sites. The interplay between the light and heavy chains (HCs) in the membrane-bound state is critical for the biological efficiency of FVIII. Here, we present our cryo-electron microscopy (EM) and structure analysis studies of human FVIII-LC, when helically assembled onto negatively charged single lipid bilayer nanotubes. The resolved FVIII-LC membrane-bound structure supports aspects of our previously proposed FVIII structure from membrane-bound two-dimensional (2D) crystals, such as only the C2 domain interacts directly with the membrane. The LC is oriented differently in the FVIII membrane-bound helical and 2D crystal structures based on EM data, and the existing X-ray structures. This flexibility of the FVIII-LC domain organization in different states is discussed in the light of the FVIIIa-FIXa complex assembly and function. PMID:23616213

  9. Membrane bound O-acyltransferases and their inhibitors.

    PubMed

    Masumoto, Naoko; Lanyon-Hogg, Thomas; Rodgers, Ursula R; Konitsiotis, Antonios D; Magee, Anthony I; Tate, Edward W

    2015-04-01

    Since the identification of the membrane-bound O-acyltransferase (MBOATs) protein family in the early 2000s, three distinct members [porcupine (PORCN), hedgehog (Hh) acyltransferase (HHAT) and ghrelin O-acyltransferase (GOAT)] have been shown to acylate specific proteins or peptides. In this review, topology determination, development of assays to measure enzymatic activities and discovery of small molecule inhibitors are compared and discussed for each of these enzymes. PMID:25849925

  10. Membrane bound Indian clade C HIV-1 envelope antigen induces antibodies to diverse and conserved epitopes upon DNA prime/protein boost in rabbits.

    PubMed

    Rangasamy, Sneha Priya; Menon, Veena; Dhopeshwarkar, Priyanka; Pal, Ranajit; Vaniambadi, Kalyanaraman S; Mahalingam, Sundarasamy

    2016-05-01

    The partial success of RV144 human clinical trial demonstrated that ALVAC prime/envelope protein boost vaccine regimen may represent a promising strategy for the development of an effective HIV-1 vaccine. Our earlier study demonstrated that a trimeric HIV-1 envelope gp145 from an Indian clade C isolate elicited cross clade neutralizing antibodies primarily towards Tier 1 isolates. In the present study, we examined the immunogenicity of DNA prime/envelope protein boost vaccine in rabbits using gp160 DNA of the Indian clade C isolate with various cytoplasmic tail truncations and trimeric gp145 protein. Cytoplasmic tail mutants of gp160 exposed epitopes that reacted strongly with a number of broadly neutralizing human monoclonal antibodies against HIV-1. Overall, envelope specific titers were found to be similar in all rabbit groups with higher pseudovirus neutralization in protein only immunized rabbits. The complete linear epitope mapping of rabbit immune sera revealed strong binding to C1, C2, V3, C3 and C4 domains of gp145. Importantly, reactivity of gp41 ecto-domain peptides was observed in DNA prime/protein boost sera but not in the sera of rabbits immunized with protein alone. Moreover, membrane anchored but not soluble envelope encoding DNA immunization elicited antibodies against linear epitopes on the conserved gp41 ecto-domain. Together, these results suggest that priming with DNA encoding cytoplasmic domains of Env alters the quality of antibodies elicited following protein boost and hence may be utilized to generate protective immunity by HIV-1 vaccine. PMID:27032514

  11. Membrane-bound Dictyostelium myosin heavy chain kinase: a developmentally regulated substrate-specific member of the protein kinase C family.

    PubMed Central

    Ravid, S; Spudich, J A

    1992-01-01

    A cDNA clone corresponding to the Dictyostelium myosin heavy chain kinase (MHCK) gene was isolated using antibodies specific to the purified enzyme. Sequence analysis of the cDNA revealed that the Dictyostelium MHCK possesses all of the domains characteristic of members of the protein kinase C family. The amino-terminal region of the MHCK contains the cysteine-rich motif with an internal duplication that is present in all known protein kinase C species. This domain precedes sequences that are highly homologous to protein kinase catalytic domains. The carboxyl-terminal region contains a cluster of 23 serine and threonine residues that may represent the autophosphorylation domain of the Dictyostelium MHCK. These results, along with previous studies that indicate that this enzyme has very restrictive substrate specificity, incorporates approximately 20 mol of phosphate per mol of kinase through an autophosphorylation reaction, and is expressed only during development, suggest that the Dictyostelium MHCK is a distinct member of the protein kinase C family and imply that this kinase family, which may include members with very specific cellular functions, may be even more heterogeneous than previously thought. Images PMID:1321427

  12. Identification and characterization of the type-IVA cyclic AMP-specific phosphodiesterase RD1 as a membrane-bound protein expressed in cerebellum.

    PubMed

    Shakur, Y; Wilson, M; Pooley, L; Lobban, M; Griffiths, S L; Campbell, A M; Beattie, J; Daly, C; Houslay, M D

    1995-03-15

    An antiserum was generated against a dodecapeptide whose sequence is found at the C-terminus of a cyclic AMP (cAMP)-specific, type-IVA phosphodiesterase encoded by the rat 'dunc-like' cyclic AMP phosphodiesterase (RD1) cDNA. This antiserum identified a single approximately 73 kDa protein species upon immunoblotting of cerebellum homogenates. This species co-migrated upon SDS/PAGE with a single immunoreactive species observed in COS cells transfected with the cDNA for RD1. Native RD1 in cerebellum was found to be predominantly (approximately 93%) membrane-associated and could be found in isolated synaptosome populations, in particular those enriched in post-synaptic densities. Fractionation of lysed synaptosomes on sucrose density gradients identified RD1 as co-migrating with the plasma membrane marker 5'-nucleotidase. Laser scanning confocal and digital deconvolution immunofluorescence studies done on intact COS cells transfected with RD1 cDNA showed RD1 to be predominantly localized to plasma membranes but also associated with the Golgi apparatus and intracellular vesicles. RD1-specific antisera immunoprecipitated phosphodiesterase activity from solubilized cerebellum membranes. This activity had the characteristics expected of the type-IV cAMP phosphodiesterase RD1 in that it was cAMP specific, exhibited a low Km cAMP of 2.3 microM, high sensitivity to inhibition by 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram) (Ki approximately 0.7 microM) and was unaffected by Ca2+/calmodulin and low concentrations of cyclic GMP. The phosphodiesterase activities of RD1 solubilized from both cerebellum and transfected COS cell membranes showed identical first-order thermal denaturation kinetics at 50 degrees C. Native RD1 from cerebellum was shown to be an integral protein in that it was solubilized using the non-ionic detergent Triton X-100 but not by either re-homogenization or high NaCl concentrations. The observation that hydroxylamine was unable to cause

  13. Membrane-Bound Dynamic Structure of an Arginine-Rich Cell-Penetrating Peptide, the Protein Transduction Domain of HIV TAT, from Solid-State NMR

    PubMed Central

    Su, Yongchao; Waring, Alan J.; Ruchala, Piotr; Hong, Mei

    2010-01-01

    The protein transduction domain of HIV-1 TAT, TAT(48-60), is an efficient cell-penetrating peptide (CPP) that diffuses across the lipid membranes of cells despite eight cationic Arg and Lys residues. To understand its mechanism of membrane translocation against the free energy barrier, we have conducted solid-state NMR experiments to determine the site-specific conformation, dynamics, and lipid interaction of the TAT peptide in anionic lipid bilayers. We found that TAT(48-60) is a highly dynamic and nearly random-coil peptide in the lipid bilayer, and inserts into the membrane-water interface near the glycerol backbone region. Arg-phosphate salt bridge interaction was revealed by short guanidinium-phosphate distances and restricted dynamics of the guanidinium. Together with the observation of strong peptide-water cross peaks in 1H spin diffusion spectra, these results indicate that TAT binding to the membrane-water interface is stabilized not only by electrostatic attraction to the anionic lipids, but also by intermolecular hydrogen bonding with the lipid phosphates and water, which may take the role of intramolecular hydrogen bonds in canonical secondary structures. The random-coil structure of TAT and another CPP, penetratin, suggests that the lack of amphipathic structure is essential for rapid translocation of these Arg-rich CPPs across the lipid membrane without causing permanent damages to the membrane integrity. PMID:20550193

  14. Dual function of membrane-bound heat shock protein 70 (Hsp70), Bag-4, and Hsp40: protection against radiation-induced effects and target structure for natural killer cells.

    PubMed

    Gehrmann, M; Marienhagen, J; Eichholtz-Wirth, H; Fritz, E; Ellwart, J; Jäättelä, M; Zilch, T; Multhoff, G

    2005-01-01

    CX+/CX- and Colo+/Colo- tumor sublines with stable heat shock protein 70 (Hsp70) high and low membrane expression were generated by fluorescence activated cell sorting of the parental human colon (CX2) and pancreas (Colo357) carcinoma cell lines, using an Hsp70-specific antibody. Two-parameter flow cytometry revealed that Hsp70 colocalizes with Bag-4, also termed silencer of death domain, not only in the cytosol but also on the plasma membrane. After nonlethal gamma-irradiation, the percentage of membrane-positive cells and the protein density of Hsp70 and Bag-4 were found to be strongly upregulated in carcinoma sublines with initially low expression levels (CX-, Colo-). Membrane expression of Hsp70 was also elevated in Bag-4 overexpressing HeLa cervix carcinoma cells when compared to neo-transfected cells. In response to gamma-irradiation, neo-transfected HeLa cells behaved like Hsp70/Bag-4 low-expressing CX- and Colo-, and Bag-4-transfected HeLa cells like Hsp70/Bag-4 high-expressing carcinoma sublines CX+ and Colo+. Immunoprecipitation studies further confirmed colocalization of Hsp70 and Bag-4 but also point to an association of Hsp70 and Hsp40 on the plasma membrane of CX+ and Colo+ cells; on CX- and Colo- tumor sublines, Hsp40 was detectable in the absence of Hsp70 and Bag-4. Other co-chaperones including Hsp60 and Hsp90 were neither found on the cell surface of CX+/CX-, Colo+/Colo- nor on HeLa neo-/HeLa Bag-4-transfected tumor cells. Functionally, Hsp70/Bag-4 and Hsp70/Hsp40 membrane-positive tumor cells appeared to be better protected against radiation-induced effects, including G2/M arrest and growth inhibition, on the one hand. On the other hand, membrane-bound Hsp70, but neither Bag-4 nor Hsp40, served as a recognition site for the cytolytic attack mediated by natural killer cells. PMID:15592361

  15. Conformational phases of membrane bound cytoskeletal filaments

    NASA Astrophysics Data System (ADS)

    Quint, David A.; Grason, Gregory; Gopinathan, Ajay

    2013-03-01

    Membrane bound cytoskeletal filaments found in living cells are employed to carry out many types of activities including cellular division, rigidity and transport. When these biopolymers are bound to a membrane surface they may take on highly non-trivial conformations as compared to when they are not bound. This leads to the natural question; What are the important interactions which drive these polymers to particular conformations when they are bound to a surface? Assuming that there are binding domains along the polymer which follow a periodic helical structure set by the natural monomeric handedness, these bound conformations must arise from the interplay of the intrinsic monomeric helicity and membrane binding. To probe this question, we study a continuous model of an elastic filament with intrinsic helicity and map out the conformational phases of this filament for various mechanical and structural parameters in our model, such as elastic stiffness and intrinsic twist of the filament. Our model allows us to gain insight into the possible mechanisms which drive real biopolymers such as actin and tubulin in eukaryotes and their prokaryotic cousins MreB and FtsZ to take on their functional conformations within living cells.

  16. Co-Immunoprecipitation of Membrane-Bound Receptors

    PubMed Central

    Avila, Julian R.; Lee, Jin Suk; Torii, Keiko U.

    2015-01-01

    The study of cell-surface receptor dynamics is critical for understanding how cells sense and respond to changing environments. Therefore, elucidating the mechanisms by which signals are perceived and communicated into the cell is necessary to understand immunity, development, and stress. Challenges in testing interactions of membrane-bound proteins include their dynamic nature, their abundance, and the complex dual environment (lipid/soluble) in which they reside. Co-Immunoprecipitation (Co-IP) of tagged membrane proteins is a widely used approach to test protein-protein interaction in vivo. In this protocol we present a method to perform Co-IP using enriched membrane proteins in isolated microsomal fractions. The different variations of this protocol are highlighted, including recommendations and troubleshooting guides in order to optimize its application. This Co-IP protocol has been developed to test the interaction of receptor-like kinases, their interacting partners, and peptide ligands in stable Arabidopsis thaliana lines, but can be modified to test interactions in transiently expressed proteins in tobacco, and potentially in other plant models, or scaled for large-scale protein-protein interactions at the membrane. PMID:26097438

  17. Platelets induce apoptosis via membrane-bound FasL

    PubMed Central

    Schleicher, Rebecca I.; Reichenbach, Frank; Kraft, Peter; Kumar, Anil; Lescan, Mario; Todt, Franziska; Göbel, Kerstin; Hilgendorf, Ingo; Geisler, Tobias; Bauer, Axel; Olbrich, Marcus; Schaller, Martin; Wesselborg, Sebastian; O’Reilly, Lorraine; Meuth, Sven G.; Schulze-Osthoff, Klaus; Gawaz, Meinrad; Li, Xuri; Kleinschnitz, Christoph; Edlich, Frank

    2015-01-01

    After tissue injury, both wound sealing and apoptosis contribute to restoration of tissue integrity and functionality. Although the role of platelets (PLTs) for wound closure and induction of regenerative processes is well established, the knowledge about their contribution to apoptosis is incomplete. Here, we show that PLTs present the death receptor Fas ligand (FasL) on their surface after activation. Activated PLTs as well as the isolated membrane fraction of activated PLTs but not of resting PLTs induced apoptosis in a dose-dependent manner in primary murine neuronal cells, human neuroblastoma cells, and mouse embryonic fibroblasts. Membrane protein from PLTs lacking membrane-bound FasL (FasL△m/△m) failed to induce apoptosis. Bax/Bak-mediated mitochondrial apoptosis signaling in target cells was not required for PLT-induced cell death, but increased the apoptotic response to PLT-induced Fas signaling. In vivo, PLT depletion significantly reduced apoptosis in a stroke model and an inflammation-independent model of N-methyl-d-aspartic acid-induced retinal apoptosis. Furthermore, experiments using PLT-specific PF4Cre+ FasLfl/fl mice demonstrated a role of PLT-derived FasL for tissue apoptosis. Because apoptosis secondary to injury prevents inflammation, our findings describe a novel mechanism on how PLTs contribute to tissue homeostasis. PMID:26232171

  18. Biochemical and molecular characterization of mitochondrial membrane-bound arginase in Heteropneustes fossilis.

    PubMed

    Mishra, Suman; Mishra, Rajnikant

    2016-05-01

    The two predominant forms of arginase, cytosolic Arginase-I and mitochondrial Arginase-II, catalyze hydrolysis of arginine into ornithine and urea. Based on presence of arginase activity in extracts using potassium chloride (KCl), mitochondrial membrane-bound arginase has also been suggested. However, the activity of arginase in fractions obtained after KCl-treatment may be either due to leakage of mitochondrial arginase or release of adhered cytosolic arginase to cell organelles having altered net charge. Therefore, it has been intended to analyse impact of KCl on ultra-structural properties of mitochondria, and biochemical analysis of mitochondrial membrane-bound proteins and arginase of Heteropneustes fossilis. Liver of H. fossilis was used for isolating mitochondria for analysis of ultrastructural properties, preparing cytosolic, mitochondrial, and mitochondrial-membrane bound extracts after treatment of KCl. Extracts were analysed for arginase activity assay, protein profiling through SDS-PAGE and MALDI MS/MS. The KCl-mediated modulation in polypeptides and arginase were also evaluated by PANTHER, MitoProt and IPSORT servers. The effects of KCl on ultra-structural integrity of mitochondria, activity of arginase, modulation on mitochondrial proteins and enzymes including arginase were observed. The 48 kDa polypeptide of mitochondrial fraction, that showed KCl-dependent alteration matched with Myb binding protein and 30 kDa bands resembles to arginase after MALDI MS/MS analysis. Results indicate KCl-dependent ultrastructural changes in mitochondria and release of mitochondrial arginase. The proposed membrane bound mitochondrial arginase could be mitochondrial arginase-II or altered form of cytosolic arginase-I contributing to KCl-induced arginase activity in H. fossilis. PMID:26922180

  19. Structure and Dynamics of the Membrane-Bound Cytochrome P450 2C9

    SciTech Connect

    Cojocaru, Vlad; Balali-Mood, Kia; Sansom, Mark S.; Wade, Rebecca C.

    2011-08-11

    The microsomal, membrane-bound, human cytochrome P450 (CYP) 2C9 is a liver-specific monooxygenase essential for drug metabolism. CYPs require electron transfer from the membrane-bound CYP reductase (CPR) for catalysis. The structural details and functional relevance of the CYP-membrane interaction are not understood. From multiple coarse grained molecular simulations started with arbitrary configurations of protein-membrane complexes, we found two predominant orientations of CYP2C9 in the membrane, both consistent with experiments and conserved in atomic-resolution simulations. The dynamics of membrane-bound and soluble CYP2C9 revealed correlations between opening and closing of different tunnels from the enzyme’s buried active site. The membrane facilitated the opening of a tunnel leading into it by stabilizing the open state of an internal aromatic gate. Other tunnels opened selectively in the simulations of product-bound CYP2C9. We propose that the membrane promotes binding of liposoluble substrates by stabilizing protein conformations with an open access tunnel and provide evidence for selective substrate access and product release routes in mammalian CYPs. The models derived here are suitable for extension to incorporate other CYPs for oligomerization studies or the CYP reductase for studies of the electron transfer mechanism, whereas the modeling procedure is generally applicable to study proteins anchored in the bilayer by a single transmembrane helix.

  20. Interfacial enzyme kinetics of a membrane bound kinase analyzed by real-time MAS-NMR.

    PubMed

    Ullrich, Sandra J; Hellmich, Ute A; Ullrich, Stefan; Glaubitz, Clemens

    2011-05-01

    The simultaneous observation of interdependent reactions within different phases as catalyzed by membrane-bound enzymes is still a challenging task. One such enzyme, the Escherichia coli integral membrane protein diacylglycerol kinase (DGK), is a key player in lipid regulation. It catalyzes the generation of phosphatidic acid within the membrane through the transfer of the γ-phosphate from soluble MgATP to membrane-bound diacylglycerol. We demonstrate that time-resolved (31)P magic angle spinning NMR offers a unique opportunity to simultaneously and directly detect both ATP hydrolysis and diacylglycerol phosphorylation. This experiment demonstrates that solid-state NMR provides a general approach for the kinetic analysis of coupled reactions at the membrane interface regardless of their compartmentalization. The enzymatic activity of DGK was probed with different lipid substrates as well as ATP analogs. Our data yield conclusions about intersubunit cooperativity, reaction stoichiometries and phosphoryl transfer mechanism and are discussed in the context of known structural data. PMID:21423170

  1. Myosin motors fragment and compact membrane-bound actin filaments

    PubMed Central

    Vogel, Sven K; Petrasek, Zdenek; Heinemann, Fabian; Schwille, Petra

    2013-01-01

    Cell cortex remodeling during cell division is a result of myofilament-driven contractility of the cortical membrane-bound actin meshwork. Little is known about the interaction between individual myofilaments and membrane-bound actin filaments. Here we reconstituted a minimal actin cortex to directly visualize the action of individual myofilaments on membrane-bound actin filaments using TIRF microscopy. We show that synthetic myofilaments fragment and compact membrane-bound actin while processively moving along actin filaments. We propose a mechanism by which tension builds up between the ends of myofilaments, resulting in compressive stress exerted to single actin filaments, causing their buckling and breakage. Modeling of this mechanism revealed that sufficient force (∼20 pN) can be generated by single myofilaments to buckle and break actin filaments. This mechanism of filament fragmentation and compaction may contribute to actin turnover and cortex reorganization during cytokinesis. DOI: http://dx.doi.org/10.7554/eLife.00116.001 PMID:23326639

  2. KCl-Dependent Release of Mitochondrial Membrane-Bound Arginase Appears to Be a Novel Variant of Arginase-II.

    PubMed

    Suman, Mishra; Rajnikant, Mishra

    2016-01-01

    Arginase regulates arginine metabolism, ornithine-urea cycle, and immunological surveillance. Arginase-I is predominant in cytosol, and arginase-II is localised in the mitochondria. A mitochondrial membrane-bound arginase has also been proposed to be adsorbed with outer membrane of mitochondria which gets released by 150 mM potassium chloride (KCl). It is presumed that inclusion of 150 mM KCl in the homogenization medium would not only facilitate release of arginase bound with outer membrane of mitochondria but also affect functional anatomy of mitochondria, mitochondrial enzymes, and proteins. Therefore, it has been intended to characterize KCl-dependent release of mitochondrial membrane-bound arginase from liver of mice. Results provide advancement in the area of arginase biology and suggest that fraction of mitochondrial membrane-bound arginase contains mitochondrial arginase-II and a variant of arginase-II. PMID:27293971

  3. KCl-Dependent Release of Mitochondrial Membrane-Bound Arginase Appears to Be a Novel Variant of Arginase-II

    PubMed Central

    Suman, Mishra; Rajnikant, Mishra

    2016-01-01

    Arginase regulates arginine metabolism, ornithine-urea cycle, and immunological surveillance. Arginase-I is predominant in cytosol, and arginase-II is localised in the mitochondria. A mitochondrial membrane-bound arginase has also been proposed to be adsorbed with outer membrane of mitochondria which gets released by 150 mM potassium chloride (KCl). It is presumed that inclusion of 150 mM KCl in the homogenization medium would not only facilitate release of arginase bound with outer membrane of mitochondria but also affect functional anatomy of mitochondria, mitochondrial enzymes, and proteins. Therefore, it has been intended to characterize KCl-dependent release of mitochondrial membrane-bound arginase from liver of mice. Results provide advancement in the area of arginase biology and suggest that fraction of mitochondrial membrane-bound arginase contains mitochondrial arginase-II and a variant of arginase-II. PMID:27293971

  4. Coordination of Copper to the Membrane-Bound Form of α-Synuclein

    SciTech Connect

    Dudzik, Christopher G.; Walter, Eric D.; Abrams, Benjamin S.; Jurica, Melissa S.; Millhauser, Glenn L.

    2013-01-01

    Aggregation of the 140 amino acid protein α-synuclein (α-syn) is linked to the development of Parkinson's disease (PD). α-Syn is a copper binding protein with potential function as a regulator of metal dependent redox activity. Epidemiological studies suggest that human exposure to excess copper increases the incidence of PD. α-Syn exists in both solution and membrane bound forms. Previous work evaluated the Cu2+ uptake for α-syn in solution and identified Met1-Asp2 and His50 as primary contributors to the coordination shell, with a dissociation constant of approximately 0.1 nM. When bound to the membrane bilayer, α-syn takes on a predominantly helical conformation, which spatially separates His50 from the protein N-terminus and is therefore incompatible with the copper coordination geometry of the solution state. Here we use circular dichroism and electron paramagnetic resonance (continuous wave and pulsed) to evaluate copper coordination to the membrane bound form of α-syn. In this molecular environment, Cu2+ binds exclusively to the protein N-terminus (Met1-Asp2) with no participation from His50. Copper does not alter the membrane bound α-syn conformation, or enhance the protein's release from the bilayer. The Cu2+ affinity is similar to that identified for solution α-syn suggesting that copper coordination is retained in the membrane. Consideration of these results suggests that copper exerts its greatest conformational affect on the solution form of α-syn and this species may therefore be precursor to PD arising from environmental copper exposure.

  5. Bacillus anthracis pXO1 plasmid encodes a putative membrane-bound bacteriocin

    PubMed Central

    Perlińska, Agata

    2014-01-01

    Evolutionary advantages over cousin cells in bacterial pathogens may decide about the success of a specific cell in its environment. Bacteria use a plethora of methods to defend against other cells and many devices to attack their opponents when competing for resources. Bacteriocins are antibacterial proteins that are used to eliminate competition. We report the discovery of a putative membrane-bound bacteriocin encoded by the Bacillus anthracis pathogenic pXO1 plasmid. We analyze the genomic structure of the bacteriocin operon. The proposed mechanisms of action predestine this operon as a potent competitive advantage over cohabitants of the same niche. PMID:25426338

  6. The maturation-inducing hormone 17a-20b-dihydroxy-4pregnen-3-one regulates gene expression of inhibin A and bambi (bone morphogenetic protein and activin membrane bound inhibitor) in the rainbow trout ovary

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transforming growth factor-beta (TGFb) superfamily members are important paracrine and autocrine regulators of ovarian development and steroidogenesis in mammals and birds, but their reproductive roles in fish are not well understood. The activin system, Tgfb, and bone morphogenetic protein 15 (Bmp...

  7. Coupled Segmentation of Nuclear and Membrane-bound Macromolecules through Voting and Multiphase Level Set

    PubMed Central

    Wen, Quan

    2014-01-01

    Membrane-bound macromolecules play an important role in tissue architecture and cell-cell communication, and is regulated by almost one-third of the genome. At the optical scale, one group of membrane proteins expresses themselves as linear structures along the cell surface boundaries, while others are sequestered; and this paper targets the former group. Segmentation of these membrane proteins on a cell-by-cell basis enables the quantitative assessment of localization for comparative analysis. However, such membrane proteins typically lack continuity, and their intensity distributions are often very heterogeneous; moreover, nuclei can form large clump, which further impedes the quantification of membrane signals on a cell-by-cell basis. To tackle these problems, we introduce a three-step process to (i) regularize the membrane signal through iterative tangential voting, (ii) constrain the location of surface proteins by nuclear features, where clumps of nuclei are segmented through a delaunay triangulation approach, and (iii) assign membrane-bound macromolecules to individual cells through an application of multi-phase geodesic level-set. We have validated our method using both synthetic data and a dataset of 200 images, and are able to demonstrate the efficacy of our approach with superior performance. PMID:25530633

  8. A multiscale approach to modelling drug metabolism by membrane-bound cytochrome P450 enzymes.

    PubMed

    Lonsdale, Richard; Rouse, Sarah L; Sansom, Mark S P; Mulholland, Adrian J

    2014-07-01

    Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes. PMID:25033460

  9. A Multiscale Approach to Modelling Drug Metabolism by Membrane-Bound Cytochrome P450 Enzymes

    PubMed Central

    Sansom, Mark S. P.; Mulholland, Adrian J.

    2014-01-01

    Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes. PMID:25033460

  10. Shortening spin-lattice relaxation using a copper-chelated lipid at low-temperatures - A magic angle spinning solid-state NMR study on a membrane-bound protein.

    PubMed

    Yamamoto, Kazutoshi; Caporini, Marc A; Im, Sangchoul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2013-12-01

    Inherent low sensitivity of NMR spectroscopy has been a major disadvantage, especially to study biomolecules like membrane proteins. Recent studies have successfully demonstrated the advantages of performing solid-state NMR experiments at very low and ultralow temperatures to enhance the sensitivity. However, the long spin-lattice relaxation time, T1, at very low temperatures is a major limitation. To overcome this difficulty, we demonstrate the use of a copper-chelated lipid for magic angle spinning solid-state NMR measurements on cytochrome-b5 reconstituted in multilamellar vesicles. Our results on multilamellar vesicles containing as small as 0.5mol% of a copper-chelated lipid can significantly shorten T1 of protons, which can be used to considerably reduce the data collection time or to enhance the signal-to-noise ratio. We also monitored the effect of slow cooling on the resolution and sensitivity of (13)C and (15)N signals from the protein and (13)C signals from lipids. PMID:24246881

  11. Shortening Spin-lattice Relaxation Using a Copper-Chelated lipid at Low-Temperatures – A Magic Angle Spinning Solid-State NMR Study on a Membrane-Bound Protein

    PubMed Central

    Yamamoto, Kazutoshi; Caporini, Marc; Im, Sangchoul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2013-01-01

    Inherent low sensitivity of NMR spectroscopy has been a major disadvantage, especially to study biomolecules like membrane proteins. Recent studies have successfully demonstrated the advantages of performing solid-state NMR experiments at very low and ultralow temperatures to enhance the sensitivity. However, the long spin-lattice relaxation time, T1, at very low temperatures is a major limitation. To overcome this difficulty, we demonstrate the use of a copper-chelated lipid for magic angle spinning solid-state NMR measurements on cytochrome-b5 reconstituted in multilamellar vesicles. Our results on multilamellar vesicles containing as small as 0.5 mole % of a copper-chelated lipid can significantly shorten T1 of protons, which can be used to considerably reduce the data collection time or to enhance the signal-to-noise ratio. We also monitored the effect of slow cooling on the resolution and sensitivity of 13C and 15N signals from the protein and 13C signals from lipids. PMID:24246881

  12. Regulatory effects of polyamines on membrane-bound acetylcholinesterase

    PubMed Central

    Kossorotow, A.; Wolf, H. U.; Seiler, N.

    1974-01-01

    The effects of putrescene, spermidine and spermine on membrane-bound acetylcholinesterase from human erythrocyte `ghosts' and the solubilized enzyme of the electric organ of the electric eel were studied by kinetic methods. Measurements were made by using a photometric method which made it possible to record the enzyme reaction in the steady-state phase. Substrate-concentration-dependent activation and inhibition of acetylcholinesterase by polyamines is similar to that by Na+, K+, Ca2+, Mg2+ and certain quaternary and bisquaternary amines. The kinetics suggest an allosteric reaction mechanism. On the basis of the kinetic results a role for the polyamines as modulators of synaptic acetylcholinesterase is proposed. PMID:4462573

  13. Membrane-bound α-synuclein interacts with glucocerebrosidase and inhibits enzyme activity

    PubMed Central

    Yap, Thai Leong; Velayati, Arash; Sidransky, Ellen; Lee, Jennifer C.

    2012-01-01

    Mutations in GBA, the gene encoding glucocerebrosidase, the lysosomal enzyme deficient in Gaucher disease increase the risk for developing Parkinson disease. Recent research suggests a relationship between glucocerebrosidase and the Parkinson disease-related amyloid-forming protein, α-synuclein; however, the specific molecular mechanisms responsible for association remain elusive. Previously, we showed that α-synuclein and glucocerebrosidase interact selectively under lysosomal conditions, and proposed that this newly identified interaction might influence cellular levels of α-synuclein by either promoting protein degradation and/or preventing aggregation. Here, we demonstrate that membrane-bound α-synuclein interacts with glucocerebrosidase, and that this complex formation inhibits enzyme function. Using site-specific fluorescence and Förster energy transfer probes, we mapped the protein-enzyme interacting regions on unilamellar vesicles. Our data suggest that on the membrane surface, the glucocerebrosidase-α-synuclein interaction involves a larger α-synuclein region compared to that found in solution. In addition, α-synuclein acts as a mixed inhibitor with an apparent IC50 in the submicromolar range. Importantly, the membrane-bound, α-helical form of α-synuclein is necessary for inhibition. This glucocerebrosidase interaction and inhibition likely contribute to the mechanism underlying GBA-associated parkinsonism. PMID:23266198

  14. Signal-Dependent Phosphorylation of the Membrane-Bound NarX Two-Component Sensor-Transmitter Protein of Escherichia coli: Nitrate Elicits a Superior Anion Ligand Response Compared to Nitrite

    PubMed Central

    Lee, Angela I.; Delgado, Asunción; Gunsalus, Robert P.

    1999-01-01

    The Nar two-component regulatory system, consisting of the dual sensor-transmitters NarX and NarQ and the dual response regulators NarL and NarP, controls the expression of various anaerobic respiratory pathway genes and fermentation pathway genes. Although both NarX and NarQ are known to detect the two environmental signals nitrate and nitrite, little is known regarding the sensitivity and selectivity of ligand for detection or activation of the sensor-transmitters. In this study, we have developed a sensitive anion-specific in vitro assay for NarX autophosphorylation by using Escherichia coli membranes highly enriched in the full-length NarX protein. In this ATP- and magnesium-dependent reaction, nitrate elicited a greater signal output (i.e., NarX autophosphorylation) than did nitrite. Nitrate stimulation occurred at concentrations as low as 5 μM, and the half-maximal level of NarX autophosphorylation occurred at approximately 35 μM nitrate. In contrast, nitrite-dependent stimulation was detected only at 500 μM, while 3.5 mM nitrite was needed to achieve half-maximal NarX autophosphorylation. Maximal nitrate- and nitrite-stimulated levels of NarX phosphorylation were five and two times, respectively, over the basal level of NarX autophosphorylation. The presence of Triton X-100 eliminated the nitrate-stimulated kinase activity and lowered the basal level of activity, suggesting that the membrane environment plays a crucial role in nitrate detection and/or regulation of kinase activity. These results provide in vitro evidence for the differential detection of dual signaling ligands by the NarX sensor-transmitter protein, which modulates the cytoplasmic NarX autokinase activity and phosphotransfer to NarL, the cognate response regulator. PMID:10464202

  15. Tunable Tensor Voting Improves Grouping of Membrane-Bound Macromolecules

    SciTech Connect

    Loss, Leandro A.; Bebis, George; Parvin, Bahram

    2009-04-15

    Membrane-bound macromolecules are responsible for structural support and mediation of cell-cell adhesion in tissues. Quantitative analysis of these macromolecules provides morphological indices for damage or loss of tissue, for example as a result of exogenous stimuli. From an optical point of view, a membrane signal may have nonuniform intensity around the cell boundary, be punctate or diffused, and may even be perceptual at certain locations along the boundary. In this paper, a method for the detection and grouping of punctate, diffuse curvilinear signals is proposed. Our work builds upon the tensor voting and the iterative voting frameworks to propose an efficient method to detect and refine perceptually interesting curvilinear structures in images. The novelty of our method lies on the idea of iteratively tuning the tensor voting fields, which allows the concentration of the votes only over areas of interest. We validate the utility of our system with synthetic and annotated real data. The effectiveness of the tunable tensor voting is demonstrated on complex phenotypic signals that are representative of membrane-bound macromolecular structures.

  16. Membrane-bound NAC transcription factors in maize and their contribution to the oxidative stress response.

    PubMed

    Wang, Dexin; Yu, Yanchong; Liu, Zhenhua; Li, Shuo; Wang, Zeli; Xiang, Fengning

    2016-09-01

    NAC membrane-bound transcription factors (NTM1-like, NTL proteins) participate in the regulation of plant development and the abiotic stress response. While their function has been thoroughly explored in Arabidopsis thaliana, this is not the case in maize. Seven ZmNTL genes were identified by an in silico scan of relevant genome sequence. All seven included a NAC domain at their N terminus, and an α-helical membrane-bound structure domain in their C terminal region. Based on their gene structure and content of conserved motifs, the seven sequences were distributed into four clades. Six of the seven ZmNTLs were associated with the plasma membrane, and the remaining one with the endoplasmic reticulum. ZmNTL2-7 were more strongly transcribed in the stem than in either the leaf or root, while ZmNTL1 transcript abundance was highest in the leaf. When the plants were exposed to either abscisic acid or hydrogen peroxide treatment, all seven genes were up-regulated in the root and stem and down-regulated in the leaf. The heterologous expression of ZmNTL1-ΔTM, 2-ΔTM and 5-ΔTM in A. thaliana reduced the level of sensitivity of the plant to hydrogen peroxide. PMID:27457981

  17. Viewing Membrane-Bound Molecular Umbrellas By Parallax Analyses

    PubMed Central

    Kondo, Masaharu; Mehiri, Mohamed

    2010-01-01

    Fluorescence quenching measurements have been made for a series of di-walled and tetra-walled molecular umbrellas having moderate (i.e., hydroxyl-) and strong (i.e., sulfate-) facial hydrophilicity, using Cascade Blue as the fluorophore. Through the use of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphotempocholine, 1-palmitoyl-2-stearoyl-(5-DOXYL)-sn-glycero-3-phosphocholine, and 1-palmitoyl-2-stearoyl-(12-DOXYL)-sn-glycero-3-phosphocholine as fluorescence quenchers, evidence has been obtained for a membrane-bound state in which the umbrella molecules lie on the surface of the lipid bilayer. In the case of the sulfated molecular umbrellas, evidence has also been obtained for a subpopulation in which the fluorophore lies deeper within the membrane. Probable structures for the shallow-lying and deep-lying molecular umbrellas are discussed. PMID:18783220

  18. A novel membrane-bound glucosyltransferase from Bradyrhizobium japonicum.

    PubMed Central

    Cohen, J L; Miller, K J

    1991-01-01

    Bacteria within the family Rhizobiaceae are distinguished by their ability to infect higher plants. The cell envelope carbohydrates of these bacteria are believed to be involved in the plant infection process. One class of cell envelope carbohydrate, the cyclic beta-1,2-glucans, is synthesized by species within two genera of this family, Agrobacterium and Rhizobium. In contrast, species of the genus Bradyrhizobium, a third genus within this family, appear to lack the capacity for cyclic beta-1,2-glucan biosynthesis. Instead, these bacteria synthesize cyclic glucans containing beta-1,6 and beta-1,3 glycosidic linkages (K.J. Miller, R.S. Gore, R. Johnson, A.J. Benesi, and V.N. Reinhold, J. Bacteriol. 172:136-142, 1990). We now report the initial characterization of a novel membrane-bound glucosyltransferase activity from Bradyrhizobium japonicum USDA 110. Analysis of the product of this glucosyltransferase activity revealed the following: the presence of beta-1,3 and beta-1,6 glycosidic linkages, an average molecular weight of 2,100, and no detectable reducing terminal residues. The glucosyltransferase activity was found to have an apparent Km of 50 microM for for UDP-glucose, and activity was stimulated optimally by Mn2+ ions. On the basis of the structural properties of the in vitro glucan product, it is possible that this membrane-bound glucosyltransferase activity may be responsible for the biosynthesis of cyclic beta-1,6-beta-1,3-glucans by this organism. PMID:1829727

  19. Localization and environment of tryptophans in soluble and membrane-bound states of a pore-forming toxin from Staphylococcus aureus.

    PubMed Central

    Raja, S M; Rawat, S S; Chattopadhyay, A; Lala, A K

    1999-01-01

    The location and environment of tryptophans in the soluble and membrane-bound forms of Staphylococcus aureus alpha-toxin were monitored using intrinsic tryptophan fluorescence. Fluorescence quenching of the toxin monomer in solution indicated varying degrees of tryptophan burial within the protein interior. N-Bromosuccinimide readily abolished 80% of the fluorescence in solution. The residual fluorescence of the modified toxin showed a blue-shifted emission maximum, a longer fluorescence lifetime as compared to the unmodified and membrane-bound alpha-toxin, and a 5- to 6-nm red edge excitation shift, all indicating a restricted tryptophan environment and deeply buried tryptophans. In the membrane-bound form, the fluorescence of alpha-toxin was quenched by iodide, indicating a conformational change leading to exposure of some tryptophans. A shorter average lifetime of tryptophans in the membrane-bound alpha-toxin as compared to the native toxin supported the conclusions based on iodide quenching of the membrane-bound toxin. Fluorescence quenching of membrane-bound alpha-toxin using brominated and spin-labeled fatty acids showed no quenching of fluorescence using brominated lipids. However, significant quenching was observed using 5- and 12-doxyl stearic acids. An average depth calculation using the parallax method indicated that the doxyl-quenchable tryptophans are located at an average depth of 10 A from the center of the bilayer close to the membrane interface. This was found to be in striking agreement with the recently described structure of the membrane-bound form of alpha-toxin. PMID:10049328

  20. Abnormal gene expression of proinflammatory cytokines and their membrane-bound receptors in the lymphocytes of depressed patients.

    PubMed

    Rizavi, Hooriyah S; Ren, Xinguo; Zhang, Hui; Bhaumik, Runa; Pandey, Ghanshyam N

    2016-06-30

    Abnormalities of protein levels of proinflammatory cytokines and their soluble receptors have been reported in plasma of depressed patients. In this study, we examined the role of cytokines and their membrane-bound receptors in major depressive disorder (MDD). We determined the protein and mRNA expression of proinflammatory cytokines, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and mRNA expression of their membrane-bound receptors in the lymphocytes from 31 hospitalized MDD patients and 30 non-hospitalized normal control (NC) subjects. The subjects were diagnosed according to DSM-IV criteria. Protein levels of cytokines were determined by ELISA, and mRNA levels in lymphocytes were determined by the qPCR method. We found that the mean mRNA levels of the proinflammatory cytokines IL-1β, IL-6, TNF-α, their receptors, TNFR1, TNFR2, IL-1R1 and the antagonist IL-1RA were significantly increased in the lymphocytes of MDD patients compared with NC. No significant differences in the lymphocyte mRNA levels of IL-1R2, IL-6R, and Gp130 were observed between MDD patients and NC. These studies suggest abnormal gene expression of these cytokines and their membrane-bound receptors in the lymphocytes of MDD patients, and that their mRNA expression levels in the lymphocytes could be a useful biomarker for depression. PMID:27138824

  1. Proinflammatory cytokines and their membrane-bound receptors are altered in the lymphocytes of schizophrenia patients

    PubMed Central

    Pandey, Ghanshyam N.; Ren, Xinguo; Rizavi, Hooriyah S.; Zhang, Hui

    2016-01-01

    Abnormalities of protein levels of proinflammatory cytokines and their soluble receptors have been reported in the plasma/serum of schizophrenia (SZ) patients. To examine if SZ is also associated with the abnormal gene expression of cytokines and their membrane-bound receptors, we studied mRNA expression of proinflammatory cytokines and their receptors in lymphocytes of SZ patients and normal control (NC) subjects. We determined the protein and mRNA expression of proinflammatory cytokines and mRNA expression of their receptors in lymphocytes from 30 SZ patients and 30 drug-free NC subjects. The subjects were diagnosed according to DSM-IV criteria. Protein levels of cytokines were determined by ELISA, and mRNA levels in lymphocytes were determined by the qPCR method. We found that the mRNA levels of IL-6, TNF-α, IL-1R1, TNFR1, and TNFR2, but not IL-1β, IL-1R2, IL-1RA, IL-6R, or GP130 were significantly increased in lymphocytes of SZ patients compared with NC subjects. We also found that the protein expression of IL-6 and TNF-α, but not IL-1β, was also significantly increased in SZ patients compared with NC subjects. These studies suggest that in addition to the reported abnormalities of proinflammatory cytokines and their soluble receptors in the plasma of SZ patients, an abnormal gene expression of these cytokines and their membrane-bound receptors may be involved in the pathogenesis of SZ. PMID:25749018

  2. Characterizing the Membrane-Bound State of Cytochrome P450 3A4: Structure, Depth of Insertion, and Orientation

    PubMed Central

    2013-01-01

    Cytochrome P450 3A4 (CYP3A4) is the most abundant membrane-associated isoform of the P450 family in humans and is responsible for biotransformation of more than 50% of drugs metabolized in the body. Despite the large number of crystallographic structures available for CYP3A4, no structural information for its membrane-bound state at an atomic level is available. In order to characterize binding, depth of insertion, membrane orientation, and lipid interactions of CYP3A4, we have employed a combined experimental and simulation approach in this study. Taking advantage of a novel membrane representation, highly mobile membrane mimetic (HMMM), with enhanced lipid mobility and dynamics, we have been able to capture spontaneous binding and insertion of the globular domain of the enzyme into the membrane in multiple independent, unbiased simulations. Despite different initial orientations and positions of the protein in solution, all the simulations converged into the same membrane-bound configuration with regard to both the depth of membrane insertion and the orientation of the enzyme on the surface of the membrane. In tandem, linear dichroism measurements performed on CYP3A4 bound to Nanodisc membranes were used to characterize the orientation of the enzyme in its membrane-bound form experimentally. The heme tilt angles measured experimentally are in close agreement with those calculated for the membrane-bound structures resulted from the simulations, thereby verifying the validity of the developed model. Membrane binding of the globular domain in CYP3A4, which appears to be independent of the presence of the transmembrane helix of the full-length enzyme, significantly reshapes the protein at the membrane interface, causing conformational changes relevant to access tunnels leading to the active site of the enzyme. PMID:23697766

  3. Hydrogen Exchange Mass Spectrometry of Functional Membrane-bound Chemotaxis Receptor Complexes

    PubMed Central

    Koshy, Seena S.; Eyles, Stephen J.; Weis, Robert M.; Thompson, Lynmarie K.

    2014-01-01

    The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (~2 Å) piston displacement of one helix of the periplasmic and transmembrane domains towards the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen exchange mass spectrometry (HDX-MS) measurements of global exchange of CF demonstrate that CF exhibits significantly slower exchange in functional complexes than in solution. Since the exchange rates in functional complexes are comparable to that of other proteins of similar structure, the CF appears to be a well-structured protein within these complexes, which is compatible with its role in propagating a signal that appears to be a tiny conformational change in the periplasmic and transmembrane domains of the receptor. We also demonstrate the feasibility of this protocol for local exchange measurements, by incorporating a pepsin digest step to produce peptides with 87% sequence coverage and only 20% back exchange. This method extends HDX-MS to membrane-bound functional complexes without detergents that may perturb the stability or structure of the system. PMID:24274333

  4. Sex steroids regulate skin pigmentation through nonclassical membrane-bound receptors.

    PubMed

    Natale, Christopher A; Duperret, Elizabeth K; Zhang, Junqian; Sadeghi, Rochelle; Dahal, Ankit; O'Brien, Kevin Tyler; Cookson, Rosa; Winkler, Jeffrey D; Ridky, Todd W

    2016-01-01

    The association between pregnancy and altered cutaneous pigmentation has been documented for over two millennia, suggesting that sex hormones play a role in regulating epidermal melanocyte (MC) homeostasis. Here we show that physiologic estrogen (17β-estradiol) and progesterone reciprocally regulate melanin synthesis. This is intriguing given that we also show that normal primary human MCs lack classical estrogen or progesterone receptors (ER or PR). Utilizing both genetic and pharmacologic approaches, we establish that sex steroid effects on human pigment synthesis are mediated by the membrane-bound, steroid hormone receptors G protein-coupled estrogen receptor (GPER), and progestin and adipoQ receptor 7 (PAQR7). Activity of these receptors was activated or inhibited by synthetic estrogen or progesterone analogs that do not bind to ER or PR. As safe and effective treatment options for skin pigmentation disorders are limited, these specific GPER and PAQR7 ligands may represent a novel class of therapeutics. PMID:27115344

  5. Dynamics of a membrane-bound tryptophan analog in environments of varying hydration: a fluorescence approach.

    PubMed

    Chattopadhyay, Amitabha; Arora, Ajuna; Kelkar, Devaki A

    2005-12-01

    Tryptophan octyl ester (TOE) represents an important model for membrane-bound tryptophan residues. In this article, we have employed a combination of wavelength-selective fluorescence and time-resolved fluorescence spectroscopies to monitor the effect of varying degrees of hydration on the dynamics of TOE in reverse micellar environments formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. Our results show that TOE exhibits red edge excitation shift (REES) and other wavelength-selective fluorescence effects when bound to reverse micelles of AOT. Fluorescence parameters such as intensity, emission maximum, anisotropy, and lifetime of TOE in reverse micelles of AOT depend on [water]/[surfactant] molar ratio (w (o)). These results are relevant and potentially useful for analyzing dynamics of proteins or peptides bound to membranes or membrane-mimetic media under conditions of changing hydration. PMID:16184387

  6. ESTRADIOL-INDUCED ENHANCEMENT OF OBJECT MEMORY CONSOLIDATION INVOLVES HIPPOCAMPAL ERK ACTIVATION AND MEMBRANE-BOUND ESTROGEN RECEPTORS

    PubMed Central

    Fernandez, Stephanie M.; Lewis, Michael C.; Pechenino, Angela S.; Harburger, Lauren L.; Orr, Patrick T.; Gresack, Jodi E.; Schafe, Glenn E.; Frick, Karyn M.

    2009-01-01

    The extracellular signal-regulated kinase (ERK) pathway is critical for various forms of learning and memory, and is activated by the potent estrogen, 17β-estradiol (E2). Here, we asked whether E2 modulates memory via ERK activation and putative membrane-bound estrogen receptors (ERs). Using ovariectomized mice, we first demonstrate that intraperitoneal (i.p.) injection of 0.2 mg/kg E2 significantly increases dorsal hippocampal levels of phosphorylated ERK protein 1 hour after injection. Second, we show that E2 administered i.p. (0.2 mg/kg) or via intrahippocampal infusion (5.0 μg/side) immediately after training in an object recognition task significantly enhances memory retention, and that the beneficial effect of i.p. E2 is blocked by dorsal hippocampal inhibition of ERK activation. Third, using bovine serum albumin-conjugated 17β-estradiol (BSA-E2), we demonstrate that E2 binding at membrane-bound ERs can increase dorsal hippocampal ERK activation and enhance object memory consolidation in an ERK-dependent manner. Fourth, we show that this effect is independent of nuclear ERs, but is dependent on the dorsal hippocampus. By demonstrating that E2 enhances memory consolidation via dorsal hippocampal ERK activation, this study is the first to identify a specific molecular pathway by which E2 modulates memory and to demonstrate a novel role for membrane-bound ERs in mediating E2-induced improvements in hippocampal memory consolidation. PMID:18753366

  7. Determining the Orientation and Localization of Membrane-Bound Peptides

    PubMed Central

    Hohlweg, Walter; Kosol, Simone; Zangger, Klaus

    2012-01-01

    Many naturally occurring bioactive peptides bind to biological membranes. Studying and elucidating the mode of interaction is often an essential step to understand their molecular and biological functions. To obtain the complete orientation and immersion depth of such compounds in the membrane or a membrane-mimetic system, a number of methods are available, which are separated in this review into four main classes: solution NMR, solid-state NMR, EPR and other methods. Solution NMR methods include the Nuclear Overhauser Effect (NOE) between peptide and membrane signals, residual dipolar couplings and the use of paramagnetic probes, either within the membrane-mimetic or in the solvent. The vast array of solid state NMR methods to study membrane-bound peptide orientation and localization includes the anisotropic chemical shift, PISA wheels, dipolar waves, the GALA, MAOS and REDOR methods and again the use of paramagnetic additives on relaxation rates. Paramagnetic additives, with their effect on spectral linewidths, have also been used in EPR spectroscopy. Additionally, the orientation of a peptide within a membrane can be obtained by the anisotropic hyperfine tensor of a rigidly attached nitroxide label. Besides these magnetic resonance techniques a series of other methods to probe the orientation of peptides in membranes has been developed, consisting of fluorescence-, infrared- and oriented circular dichroism spectroscopy, colorimetry, interface-sensitive X-ray and neutron scattering and Quartz crystal microbalance. PMID:22044140

  8. Membrane-Bound Structure and Topology of a Human Alpha Defensin Indicates A Dimer Pore Mechanism for Membrane Disruption

    PubMed Central

    Zhang, Yuan; Lu, Wuyuan; Hong, Mei

    2010-01-01

    Defensins are cationic and disulfide-bonded host defense proteins of many animals that target microbial cell membranes. Elucidating the three-dimensional structure, dynamics and topology of these proteins in phospholipid bilayers is important for understanding their mechanisms of action. Using solid-state NMR spectroscopy, we have now determined the conformation, dynamics, oligomeric state and topology of a human α-defensin, HNP-1, in DMPC/DMPG bilayers. 2D correlation spectra show that membrane-bound HNP-1 exhibits a similar conformation to the water-soluble state, except for the turn connecting the β2 and β3 strands, whose sidechains exhibit immobilization and conformational perturbation upon membrane binding. At high protein/lipid ratios, rapid 1H spin diffusion from the lipid chains to the protein was observed, indicating that HNP-1 was well inserted into the hydrocarbon core of the bilayer. Arg Cζ-lipid 31P distances indicate that only one of the four Arg residues forms tight hydrogen-bonded guanidinium-phosphate complexes. The protein is predominantly dimerized at high protein/lipid molar ratios, as shown by 19F spin diffusion experiments. The presence of a small fraction of monomers and the shallower insertion at lower protein concentrations suggest that HNP-1 adopts concentration-dependent oligomerization and membrane-bound structure. These data strongly support a “dimer pore” topology of HNP-1 in which the polar top of the dimer lines an aqueous pore while the hydrophobic bottom faces the lipid chains. In this structure R25 lies closest to the membrane surface among the four Arg residues. The pore does not have large lipid disorder, in contrast to the toroidal pores formed by protegrin-1, a two-stranded β-hairpin antimicrobial peptide. These results provide the first glimpse into the membrane-bound structure and mechanism of action of human α-defensins. PMID:20961099

  9. Crystal structure of a membrane-bound l-amino acid deaminase from Proteus vulgaris.

    PubMed

    Ju, Yingchen; Tong, Shuilong; Gao, Yongxiang; Zhao, Wei; Liu, Qi; Gu, Qiong; Xu, Jun; Niu, Liwen; Teng, Maikun; Zhou, Huihao

    2016-09-01

    l-amino acid oxidases/deaminases (LAAOs/LAADs) are a class of oxidoreductases catalyzing the oxidative deamination of l-amino acids to α-keto acids. They are widely distributed in eukaryotic and prokaryotic organisms, and exhibit diverse substrate specificity, post-translational modifications and cellular localization. While LAAOs isolated from snake venom have been extensively characterized, the structures and functions of LAAOs from other species are largely unknown. Here, we reported crystal structure of a bacterial membrane-bound LAAD from Proteus vulgaris (pvLAAD) in complex with flavin adenine dinucleotide (FAD). We found that the overall fold of pvLAAD does not resemble typical LAAOs. Instead it, is similar to d-amino acid oxidases (DAAOs) with an additional hydrophobic insertion module on protein surface. Structural analysis and liposome-binding assays suggested that the hydrophobic module serves as an extra membrane-binding site for LAADs. Bacteria from genera Proteus and Providencia were found to encode two classes of membrane-bound LAADs. Based on our structure, the key roles of residues Q278 and L317 in substrate selectivity were proposed and biochemically analyzed. While LAADs on the membrane were proposed to transfer electrons to respiratory chain for FAD re-oxidization, we observed that the purified pvLAAD could generate a significant amount of hydrogen peroxide in vitro, suggesting it could use dioxygen to directly re-oxidize FADH2 as what typical LAAOs usually do. These findings provide a novel insights for a better understanding this class of enzymes and will help developing biocatalysts for industrial applications. PMID:27422658

  10. Sialic Acid Is Required for Neuronal Inhibition by Soluble MAG but not for Membrane Bound MAG.

    PubMed

    Al-Bashir, Najat; Mellado, Wilfredo; Filbin, Marie T

    2016-01-01

    Myelin-Associated Glycoprotein (MAG), a major inhibitor of axonal growth, is a member of the immunoglobulin (Ig) super-family. Importantly, MAG (also known as Siglec-4) is a member of the Siglec family of proteins (sialic acid-binding, immunoglobulin-like lectins), MAG binds to complex gangliosides, specifically GD1a and/or GT1b. Therefore, it has been proposed as neuronal receptors for MAG inhibitory effect of axonal growth. Previously, we showed that MAG binds sialic acid through domain 1 at Arg118 and is able to inhibit axonal growth through domain 5. We developed a neurite outgrowth (NOG) assay, in which both wild type MAG and mutated MAG (MAG Arg118) are expressed on cells. In addition we also developed a soluble form NOG in which we utilized soluble MAG-Fc and mutated MAG (Arg118-Fc). Only MAG-Fc is able to inhibit NOG, but not mutated MAG (Arg118)-Fc that has been mutated at its sialic acid binding site. However, both forms of membrane bound MAG- and MAG (Arg118)- expressing cells still inhibit NOG. Here, we review various results from different groups regarding MAG's inhibition of axonal growth. Also, we propose a model in which the sialic acid binding is not necessary for the inhibition induced by the membrane form of MAG, but it is necessary for the soluble form of MAG. This finding highlights the importance of understanding the different mechanisms by which MAG inhibits NOG in both the soluble fragmented form and the membrane-bound form in myelin debris following CNS damage. PMID:27065798

  11. Sialic Acid Is Required for Neuronal Inhibition by Soluble MAG but not for Membrane Bound MAG

    PubMed Central

    Al-Bashir, Najat; Mellado, Wilfredo; Filbin, Marie T.

    2016-01-01

    Myelin-Associated Glycoprotein (MAG), a major inhibitor of axonal growth, is a member of the immunoglobulin (Ig) super-family. Importantly, MAG (also known as Siglec-4) is a member of the Siglec family of proteins (sialic acid-binding, immunoglobulin-like lectins), MAG binds to complex gangliosides, specifically GD1a and/or GT1b. Therefore, it has been proposed as neuronal receptors for MAG inhibitory effect of axonal growth. Previously, we showed that MAG binds sialic acid through domain 1 at Arg118 and is able to inhibit axonal growth through domain 5. We developed a neurite outgrowth (NOG) assay, in which both wild type MAG and mutated MAG (MAG Arg118) are expressed on cells. In addition we also developed a soluble form NOG in which we utilized soluble MAG-Fc and mutated MAG (Arg118-Fc). Only MAG-Fc is able to inhibit NOG, but not mutated MAG (Arg118)-Fc that has been mutated at its sialic acid binding site. However, both forms of membrane bound MAG- and MAG (Arg118)- expressing cells still inhibit NOG. Here, we review various results from different groups regarding MAG’s inhibition of axonal growth. Also, we propose a model in which the sialic acid binding is not necessary for the inhibition induced by the membrane form of MAG, but it is necessary for the soluble form of MAG. This finding highlights the importance of understanding the different mechanisms by which MAG inhibits NOG in both the soluble fragmented form and the membrane-bound form in myelin debris following CNS damage. PMID:27065798

  12. Evolutionarily divergent, Na+-regulated H+-transporting membrane-bound pyrophosphatases.

    PubMed

    Luoto, Heidi H; Nordbo, Erika; Malinen, Anssi M; Baykov, Alexander A; Lahti, Reijo

    2015-04-15

    Membrane-bound pyrophosphatase (mPPases) of various types consume pyrophosphate (PPi) to drive active H+ or Na+ transport across membranes. H+-transporting PPases are divided into phylogenetically distinct K+-independent and K+-dependent subfamilies. In the present study, we describe a group of 46 bacterial proteins and one archaeal protein that are only distantly related to known mPPases (23%-34% sequence identity). Despite this evolutionary divergence, these proteins contain the full set of 12 polar residues that interact with PPi, the nucleophilic water and five cofactor Mg2+ ions found in 'canonical' mPPases. They also contain a specific lysine residue that confers K+ independence on canonical mPPases. Two of the proteins (from Chlorobium limicola and Cellulomonas fimi) were expressed in Escherichia coli and shown to catalyse Mg2+-dependent PPi hydrolysis coupled with electrogenic H+, but not Na+ transport, in inverted membrane vesicles. Unique features of the new H+-PPases include their inhibition by Na+ and inhibition or activation, depending on PPi concentration, by K+ ions. Kinetic analyses of PPi hydrolysis over wide ranges of cofactor (Mg2+) and substrate (Mg2-PPi) concentrations indicated that the alkali cations displace Mg2+ from the enzyme, thereby arresting substrate conversion. These data define the new proteins as a novel subfamily of H+-transporting mPPases that partly retained the Na+ and K+ regulation patterns of their precursor Na+-transporting mPPases. PMID:25662511

  13. Developmental changes in the composition of polyadenylated RNA isolated from free and membrane-bound polyribosomes of the rat forebrain, analysed by translation in vitro.

    PubMed Central

    Hall, C; Lim, L

    1981-01-01

    Free and membrane-bound polyribosomes were isolated from the rat forebrain during its development. Polyadenylated RNA [poly(A)+ RNA] was isolated from both fractions, by using oligo(dT)-cellulose chromatography, and its composition studied by translating the poly(A)+ RNA in vitro in reticulocyte lysates. Electrophoretic analysis of the translation products showed that both free and membrane-bound polyribosomal poly(A)+ RNA gave many common components, but that there were also distinct differences in the protein composition of the products of the two fractions. Several proteins, of mol.wts. 39 000, 37 000, 31 000, 27 000 and 17 000, appeared to be products predominantly of free polyribosomal poly(A)+ RNA, whereas others, of mol.wt. 47 000, 33 000, 24 000 and 21 000 were specific to the membrane-bound polyribosomal poly(A)+ RNA fraction. More developmental changes were observed in the translational products of the membrane-bound poly(A)+ RNA fraction. Proteins of mol.wts. 33 000 and 21 000, which were predominant components of the translational products of this fraction when isolated from 10-day and older rats, were not present in translational products derived from preparations isolated from 3-day-old rats. The developmental appearance of these proteins as translational products of the membrane-bound poly(A)+ RNA suggests the appearance of new mRNA species. These transcriptional changes are discussed in relation to processes involved in brain differentiation, including myelination. Images Fig. 2. Fig. 4. Fig. 5. Fig. 6. PMID:6171267

  14. Purification, characterization, and crystallization of membrane bound Escherichia coli tyrosine kinase.

    PubMed

    Chesterman, Chelsy; Jia, Zongchao

    2016-09-01

    Escherichia coli tyrosine kinase (Etk) is a membrane bound kinase in gram-negative bacteria that regulates the export of capsular polysaccharides (CPS). The molecular mechanism behind CPS regulation remains unclear, despite access to a crystal structure of the cytoplasmic kinase domain of Etk. In this study, an efficient protocol to produce full length Etk solubilized in n-dodecyl-β-d-maltoside has been established with high yield. We have determined that detergent solubilized Etk retains kinase activity, but the protein is prone to aggregation, degradation, and has been unsuccessful in protein crystallization trials. In response, we designed and characterized truncations of Etk that do not aggregate and have led to successful crystallization experiments. In this article, we discuss our optimized expression and purification protocol for Etk, the design of Etk protein truncations, and the behavior of Etk during purification in a range of stabilizing detergents. These efforts have successfully produced protein suitable for crystallization. Our results will be a useful guide for future structural and functional studies of the bacterial tyrosine kinase family. PMID:26363120

  15. [Purification and properties of membrane-bound methane hydroxylase from Methylococcus capsulatus (strain M)].

    PubMed

    Gvozdev, R I; Tukhvatullin, I A; Tumanova, L V

    2008-01-01

    Membrane fraction of Methylococcus capsulatus (strain M) were treated with [14C]acetylene, an affinity label binding to the active center of membrane-bound methane monooxygenase (MMO). High-purity particulate form of methane hydroxylase (pMH) was obtained by ion exchange and hydrophobic chromatography. According to SDS-PAGE data, the enzyme contained three polypeptides with molecular weights of 47 (alpha), 27 (beta), and 25 (gamma) kDa in the ratio 1:1:1. The radiolabel was contained in the beta-subunit of pMH. The protein contained 1 or 2 atoms of nonheme iron and 2-4 atoms of copper per a minimum molecular weight of 99 kDa. This protein did not oxidize methane or propylene in the presence of NADH but was able to oxidize low quantities of methane in the presence of duroquinol. It was established that methanol dehydrogenase (MD) and NADH oxidoreductase (NADH-OR) are peripheral membrane proteins. Possible causes of low activity of high-purity methane hydroxylase are discussed. PMID:18946992

  16. Structural Ensembles of Membrane-bound α-Synuclein Reveal the Molecular Determinants of Synaptic Vesicle Affinity

    PubMed Central

    Fusco, Giuliana; De Simone, Alfonso; Arosio, Paolo; Vendruscolo, Michele; Veglia, Gianluigi; Dobson, Christopher M.

    2016-01-01

    A detailed characterisation of the molecular determinants of membrane binding by α-synuclein (αS), a 140-residue protein whose aggregation is associated with Parkinson’s disease, is of fundamental significance to clarify the manner in which the balance between functional and dysfunctional processes are regulated for this protein. Despite its biological relevance, the structural nature of the membrane-bound state αS remains elusive, in part because of the intrinsically dynamic nature of the protein and also because of the difficulties in studying this state in a physiologically relevant environment. In the present study we have used solid-state NMR and restrained MD simulations to refine structure and topology of the N-terminal region of αS bound to the surface of synaptic-like membranes. This region has fundamental importance in the binding mechanism of αS as it acts as to anchor the protein to lipid bilayers. The results enabled the identification of the key elements for the biological properties of αS in its membrane-bound state. PMID:27273030

  17. Structural Ensembles of Membrane-bound α-Synuclein Reveal the Molecular Determinants of Synaptic Vesicle Affinity.

    PubMed

    Fusco, Giuliana; De Simone, Alfonso; Arosio, Paolo; Vendruscolo, Michele; Veglia, Gianluigi; Dobson, Christopher M

    2016-01-01

    A detailed characterisation of the molecular determinants of membrane binding by α-synuclein (αS), a 140-residue protein whose aggregation is associated with Parkinson's disease, is of fundamental significance to clarify the manner in which the balance between functional and dysfunctional processes are regulated for this protein. Despite its biological relevance, the structural nature of the membrane-bound state αS remains elusive, in part because of the intrinsically dynamic nature of the protein and also because of the difficulties in studying this state in a physiologically relevant environment. In the present study we have used solid-state NMR and restrained MD simulations to refine structure and topology of the N-terminal region of αS bound to the surface of synaptic-like membranes. This region has fundamental importance in the binding mechanism of αS as it acts as to anchor the protein to lipid bilayers. The results enabled the identification of the key elements for the biological properties of αS in its membrane-bound state. PMID:27273030

  18. Structural Features of Membrane-bound Glucocerebrosidase and α-Synuclein Probed by Neutron Reflectometry and Fluorescence Spectroscopy*

    PubMed Central

    Yap, Thai Leong; Jiang, Zhiping; Heinrich, Frank; Gruschus, James M.; Pfefferkorn, Candace M.; Barros, Marilia; Curtis, Joseph E.; Sidransky, Ellen; Lee, Jennifer C.

    2015-01-01

    Mutations in glucocerebrosidase (GCase), the enzyme deficient in Gaucher disease, are a common genetic risk factor for the development of Parkinson disease and related disorders, implicating the role of this lysosomal hydrolase in the disease etiology. A specific physical interaction exists between the Parkinson disease-related protein α-synuclein (α-syn) and GCase both in solution and on the lipid membrane, resulting in efficient enzyme inhibition. Here, neutron reflectometry was employed as a first direct structural characterization of GCase and α-syn·GCase complex on a sparsely-tethered lipid bilayer, revealing the orientation of the membrane-bound GCase. GCase binds to and partially inserts into the bilayer with its active site most likely lying just above the membrane-water interface. The interaction was further characterized by intrinsic Trp fluorescence, circular dichroism, and surface plasmon resonance spectroscopy. Both Trp fluorescence and neutron reflectometry results suggest a rearrangement of loops surrounding the catalytic site, where they extend into the hydrocarbon chain region of the outer leaflet. Taking advantage of contrasting neutron scattering length densities, the use of deuterated α-syn versus protiated GCase showed a large change in the membrane-bound structure of α-syn in the complex. We propose a model of α-syn·GCase on the membrane, providing structural insights into inhibition of GCase by α-syn. The interaction displaces GCase away from the membrane, possibly impeding substrate access and perturbing the active site. GCase greatly alters membrane-bound α-syn, moving helical residues away from the bilayer, which could impact the degradation of α-syn in the lysosome where these two proteins interact. PMID:25429104

  19. Characterization of 19 Genes Encoding Membrane-Bound Fatty Acid Desaturases and their Expression Profiles in Gossypium raimondii Under Low Temperature.

    PubMed

    Liu, Wei; Li, Wei; He, Qiuling; Daud, Muhammad Khan; Chen, Jinhong; Zhu, Shuijin

    2015-01-01

    To produce unsaturated fatty acids, membrane-bound fatty acid desaturases (FADs) can be exploited to introduce double bonds into the acyl chains of fatty acids. In this study, 19 membrane-bound FAD genes were identified in Gossypium raimondii through database searches and were classified into four different subfamilies based on phylogenetic analysis. All 19 membrane-bound FAD proteins shared three highly conserved histidine boxes, except for GrFAD2.1, which lost the third histidine box in the C-terminal region. In the G. raimondii genome, tandem duplication might have led to the increasing size of the FAD2 cluster in the Omega Desaturase subfamily, whereas segmental duplication appeared to be the dominant mechanism for the expansion of the Sphingolipid and Front-end Desaturase subfamilies. Gene expression analysis showed that seven membrane-bound FAD genes were significantly up-regulated and that five genes were greatly suppressed in G. raimondii leaves exposed to low temperature conditions. PMID:25894196

  20. Characterization of 19 Genes Encoding Membrane-Bound Fatty Acid Desaturases and their Expression Profiles in Gossypium raimondii Under Low Temperature

    PubMed Central

    He, Qiuling; Daud, Muhammad Khan; Chen, Jinhong; Zhu, Shuijin

    2015-01-01

    To produce unsaturated fatty acids, membrane-bound fatty acid desaturases (FADs) can be exploited to introduce double bonds into the acyl chains of fatty acids. In this study, 19 membrane-bound FAD genes were identified in Gossypium raimondii through database searches and were classified into four different subfamilies based on phylogenetic analysis. All 19 membrane-bound FAD proteins shared three highly conserved histidine boxes, except for GrFAD2.1, which lost the third histidine box in the C-terminal region. In the G. raimondii genome, tandem duplication might have led to the increasing size of the FAD2 cluster in the Omega Desaturase subfamily, whereas segmental duplication appeared to be the dominant mechanism for the expansion of the Sphingolipid and Front-end Desaturase subfamilies. Gene expression analysis showed that seven membrane-bound FAD genes were significantly up-regulated and that five genes were greatly suppressed in G. raimondii leaves exposed to low temperature conditions. PMID:25894196

  1. Membrane-Bound PenA β-Lactamase of Burkholderia pseudomallei.

    PubMed

    Randall, Linnell B; Dobos, Karen; Papp-Wallace, Krisztina M; Bonomo, Robert A; Schweizer, Herbert P

    2015-01-01

    Burkholderia pseudomallei is the etiologic agent of melioidosis, a difficult-to-treat disease with diverse clinical manifestations. β-Lactam antibiotics such as ceftazidime are crucial to the success of melioidosis therapy. Ceftazidime-resistant clinical isolates have been described, and the most common mechanism is point mutations affecting expression or critical amino acid residues of the chromosomally encoded class A PenA β-lactamase. We previously showed that PenA was exported via the twin arginine translocase system and associated with the spheroplast fraction. We now show that PenA is a membrane-bound lipoprotein. The protein and accompanying β-lactamase activity are found in the membrane fraction and can be extracted with Triton X-114. Treatment with globomycin of B. pseudomallei cells expressing PenA results in accumulation of the prolipoprotein. Mass spectrometric analysis of extracted membrane proteins reveals a protein peak whose mass is consistent with a triacylated PenA protein. Mutation of a crucial lipobox cysteine at position 23 to a serine residue results in loss of β-lactamase activity and absence of detectable PenAC23S protein. A concomitant isoleucine-to-alanine change at position 20 in the signal peptide processing site in the PenAC23S mutant results in a nonlipidated protein (PenAI20A C23S) that is processed by signal peptidase I and exhibits β-lactamase activity. The resistance profile of a B. pseudomallei strain expressing this protein is indistinguishable from the profile of the isogenic strain expressing wild-type PenA. The data show that PenA membrane association is not required for resistance and must serve another purpose. PMID:26711764

  2. Role of nickel in membrane-bound hydrogenase and nickel metabolism in Rhizobium japonicum

    SciTech Connect

    Stults, L.W.

    1986-01-01

    The membrane-bound hydrogenase of Rhizobium japonicum requires nickel for activity. Radioactive /sup 63/Ni co-migrates with hydrogenase activity in native gel systems and co-elutes with purified hydrogenase form an affinity matrix column. A simplified scheme for the purification of hydrogenase has been developed and constitutes the first report of the aerobic purification of this enzyme from R. japonicum. The aerobic purification utilizes the general affinity matrix. Reactive Red 120-agarose and results in higher specific activity and yield of enzyme than previously reported. The stability of aerobically purified hydrogenase to oxygen is substantially greater than that reported for anaerobically isolated enzyme. Reduction of the aerobically purified enzyme in the presence of oxygen, however, results in the rapid loss of activity. R. japonicum cells accumulate nickel during heterotrophic growth and as non-growing cells. The hydrogenase constitutive mutant SR470 accumulates substantially greater amounts of nickel under both conditions. Kinetic studies indicate that the nickel uptake system in the hydrogenase constitutive mutant SR470 is upregulated relative to SRwt cells. The uptake system is specific for nickel, although a 10-fold excess (relative to nickel) of copper or zinc inhibits nickel uptake. The nickel uptake system appears to require energy. Under nickel-free conditions hydrogenase protein is not synthesized as determined by cross-reactivity with antibodies directed against hydrogenase, indicating that nickel regulates the formation of the enzyme as well as being a constituent of the active protein.

  3. pH-induced conformational changes of membrane-bound influenza hemagglutinin and its effect on target lipid bilayers.

    PubMed Central

    Gray, C.; Tamm, L. K.

    1998-01-01

    Influenza virus hemagglutinin (HA) has served as a paradigm for both pH-dependent and -independent viral membrane fusion. Although large conformational changes were observed by X-ray crystallography when soluble fragments of HA were subjected to fusion-pH conditions, it is not clear whether the same changes occur in membrane-bound HA, what the spatial relationship is between the conformationally changed HA and the target and viral membranes, and in what way HA perturbs the target membrane at low pH. We have taken a spectroscopic approach using an array of recently developed FTIR techniques to address these questions. Difference attenuated total reflection FTIR spectroscopy was employed to reveal reversible and irreversible components of the pH-induced conformational change of the membrane-bound bromelain fragment of HA, BHA. Additional proteolytic fragments of BHA were produced which permitted a tentative assignment of the observed changes to the HA1 and HA2 subunits, respectively. The membrane-bound HA1 subunit undergoes a reversible conformational change, which most likely involves the loss of a small proportion of beta-sheet at low pH. BHA was found to undergo a partially reversible tilting motion relative to the target membrane upon exposure to pH 5, indicating a previously undescribed hinge near the anchoring point to the target membrane. Time-resolved amide H/D exchange experiments revealed a more dynamic (tertiary) structure of membrane-bound BHA and its HA2, but not its HA1, subunit. Finally BHA and, to a lesser degree, HA1 perturbed the lipid bilayer of the target membrane at the interface, as assessed by spectral changes of the lipid ester carbonyl groups. These results are discussed in the context of a complementary study of HA that was bound to viral membranes through its transmembrane peptide (Gray C, Tamm LK, 1997, Protein Sci 6:1993-2006). A distinctive role for the HA1 subunit in the conformational change of HA becomes apparent from these combined

  4. C. elegans uses Liquid-Liquid Demixing for the Assembly of Non-Membrane-Bound Compartments

    NASA Astrophysics Data System (ADS)

    Weber, Christoph A.; Juelicher, Frank; Diaz Delgadillo, Andres Felipe; Jawerth, Louise; Hyman, Anthony A.; Department Biological Physics Team; Hyman Lab Collaboration

    2015-03-01

    P granules are liquid cytoplasmic RNA/Protein condensates known to determine the germ lineage in Caenorhabditis elegans. They resemble striking similarities with liquid droplets, such as dripping, shearing and wetting. Assuming that P granules are liquid-like we consider how they form in the crowded cytoplasm. Using confocal and light-sheet microscopy, P granule formation in-vivo and in-vitro is shown to share all hallmarks with a liquid-liquid phase-separation. Specifically, demixing is determined by temperature and concentration, the droplet formation is reversible with respect to temperature quenches and there is evidence for droplet growth due to coalescence and Ostwald-ripening. Liquid-liquid demixing in-vivo breaks the paradigmatic view that a molecular machinery is necessary to build up organelles through complex biological pathways. Instead we propose that P granules form following a Flory-Huggins model. Liquid-liquid demixing could also serve as a mechanism for the assembly of non-membrane-bound compartments in other living organisms.

  5. Sex steroids regulate skin pigmentation through nonclassical membrane-bound receptors

    PubMed Central

    Natale, Christopher A; Duperret, Elizabeth K; Zhang, Junqian; Sadeghi, Rochelle; Dahal, Ankit; O'Brien, Kevin Tyler; Cookson, Rosa; Winkler, Jeffrey D; Ridky, Todd W

    2016-01-01

    The association between pregnancy and altered cutaneous pigmentation has been documented for over two millennia, suggesting that sex hormones play a role in regulating epidermal melanocyte (MC) homeostasis. Here we show that physiologic estrogen (17β-estradiol) and progesterone reciprocally regulate melanin synthesis. This is intriguing given that we also show that normal primary human MCs lack classical estrogen or progesterone receptors (ER or PR). Utilizing both genetic and pharmacologic approaches, we establish that sex steroid effects on human pigment synthesis are mediated by the membrane-bound, steroid hormone receptors G protein-coupled estrogen receptor (GPER), and progestin and adipoQ receptor 7 (PAQR7). Activity of these receptors was activated or inhibited by synthetic estrogen or progesterone analogs that do not bind to ER or PR. As safe and effective treatment options for skin pigmentation disorders are limited, these specific GPER and PAQR7 ligands may represent a novel class of therapeutics. DOI: http://dx.doi.org/10.7554/eLife.15104.001 PMID:27115344

  6. Identification of a Membrane-bound Prepore Species Clarifies the Lytic Mechanism of Actinoporins.

    PubMed

    Morante, Koldo; Bellomio, Augusto; Gil-Cartón, David; Redondo-Morata, Lorena; Sot, Jesús; Scheuring, Simon; Valle, Mikel; González-Mañas, Juan Manuel; Tsumoto, Kouhei; Caaveiro, Jose M M

    2016-09-01

    Pore-forming toxins (PFTs) are cytolytic proteins belonging to the molecular warfare apparatus of living organisms. The assembly of the functional transmembrane pore requires several intermediate steps ranging from a water-soluble monomeric species to the multimeric ensemble inserted in the cell membrane. The non-lytic oligomeric intermediate known as prepore plays an essential role in the mechanism of insertion of the class of β-PFTs. However, in the class of α-PFTs, like the actinoporins produced by sea anemones, evidence of membrane-bound prepores is still lacking. We have employed single-particle cryo-electron microscopy (cryo-EM) and atomic force microscopy to identify, for the first time, a prepore species of the actinoporin fragaceatoxin C bound to lipid vesicles. The size of the prepore coincides with that of the functional pore, except for the transmembrane region, which is absent in the prepore. Biochemical assays indicated that, in the prepore species, the N terminus is not inserted in the bilayer but is exposed to the aqueous solution. Our study reveals the structure of the prepore in actinoporins and highlights the role of structural intermediates for the formation of cytolytic pores by an α-PFT. PMID:27445331

  7. Endocytic Trafficking of Membrane-Bound Cargo: A Flotillin Point of View

    PubMed Central

    Meister, Melanie; Tikkanen, Ritva

    2014-01-01

    The ubiquitous and highly conserved flotillin proteins, flotillin-1 and flotillin-2, have been shown to be involved in various cellular processes such as cell adhesion, signal transduction through receptor tyrosine kinases as well as in cellular trafficking pathways. Due to the fact that flotillins are acylated and form hetero-oligomers, they constitutively associate with cholesterol-enriched lipid microdomains. In recent years, such microdomains have been appreciated as platforms that participate in endocytosis and other cellular trafficking steps. This review summarizes the current findings on the role of flotillins in membrane-bound cargo endocytosis and endosomal trafficking events. We will discuss the proposed function of flotillins in endocytosis in the light of recent findings that point towards a role for flotillins in a step that precedes the actual endocytic uptake of cargo molecules. Recent findings have also revealed that flotillins may be important for endosomal sorting and recycling of specific cargo molecules. In addition to these aspects, the cellular trafficking pathway of flotillins themselves as potential cargo in the context of growth factor signaling will be discussed. PMID:25019426

  8. Oligomerization of Membrane-Bound Diphtheria Toxin (CRM197) Facilitates a Transition to the Open Form and Deep Insertion

    PubMed Central

    Kent, M. S.; Yim, H.; Murton, J. K.; Satija, S.; Majewski, J.; Kuzmenko, I.

    2008-01-01

    Diphtheria toxin (DT) contains separate domains for receptor-specific binding, translocation, and enzymatic activity. After binding to cells, DT is taken up into endosome-like acidic compartments where the translocation domain inserts into the endosomal membrane and releases the catalytic domain into the cytosol. The process by which the catalytic domain is translocated across the endosomal membrane is known to involve pH-induced conformational changes; however, the molecular mechanisms are not yet understood, in large part due to the challenge of probing the conformation of the membrane-bound protein. In this work neutron reflection provided detailed conformational information for membrane-bound DT (CRM197) in situ. The data revealed that the bound toxin oligomerizes with increasing DT concentration and that the oligomeric form (and only the oligomeric form) undergoes a large extension into solution with decreasing pH that coincides with deep insertion of residues into the membrane. We interpret the large extension as a transition to the open form. These results thus indicate that as a function of bulk DT concentration, adsorbed DT passes from an inactive state with a monomeric dimension normal to the plane of the membrane to an active state with a dimeric dimension normal to the plane of the membrane. PMID:18055530

  9. Purification and properties of the membrane-bound by hydrogenase from Desulfovibrio desulfuricans.

    PubMed

    Lalla-Maharajh, W V; Hall, D O; Cammack, R; Rao, K K; Le Gall, J

    1983-02-01

    The membrane-bound hydrogenase from the anaerobic sulphate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) has been purified to homogeneity, with an overall 80-fold purification and a specific activity of 70 mumol of H2 evolved/min per mg of protein. The hydrogenase had a relative molecular mass of 58 000 as determined by gel filtration and was estimated to contain six iron atoms and six acid-labile sulphur groups per molecule. The absorption spectrum of the enzyme was characteristic of an iron-sulphur protein. The E400 and E280 were 28 500 and 109 000 M-1.cm-1 respectively. The e.s.r. of the oxidized protein indicated the presence of [4Fe-4S]3+ or [3Fe-3S]3+, and another paramagnetic centre, probably Ni(III). The hydrogenase was inhibited by heavy-metal salts, carbon monoxide and high ionic strength. However, it was resistant to inhibition by thiol-blocking and metal-complexing reagents. N-Bromosuccinimide totally inhibited the enzyme activity at low concentrations. The enzyme was stable to O2 over long periods and to high temperatures. It catalyses both H2-evolution and H2-uptake with a variety of artificial electron carriers. D. desulfuricans cytochrome C3, its natural electron carrier, had a high affinity for the enzyme (Km = 2 microns). Rate enhancement was observed when cytochrome C3 was added to Methyl Viologen in the H2-evolution assay. The pH optimum for H2-evolution was 6.5. PMID:6303306

  10. Quantum Chemical Calculations of Amide-15N Chemical Shift Anisotropy Tensors for a Membrane-Bound Cytochrome b5

    PubMed Central

    Pandey, Manoj Kumar; Ramamoorthy, Ayyalusamy

    2013-01-01

    There is considerable interest in determining amide-15N chemical shift anisotropy (CSA) tensors from biomolecules and understanding their variation for structural and dynamics studies using solution and solid-state NMR spectroscopy and also by quantum chemical calculations. Due to the difficulties associated with the measurement of CSA tensors from membrane proteins, NMR-based structural studies heavily relied on the CSA tensors determined from model systems, typically single crystals of model peptides. In the present study, the principal components of backbone amide-15N CSA tensor have been determined using density functional theory for a 16.7-kDa membrane-bound paramagnetic heme containing protein, cytochrome b5 (cytb5). All the calculations were performed by taking residues within 5Å distance from the backbone amide-15N nucleus of interest. The calculated amide-15N CSA spans agree less well with our solution NMR data determined for an effective internuclear distance rN-H = 1.023 Å and a constant angle β = 18° that the least shielded component (δ11) makes with the N-H bond. The variation of amide-15N CSA span obtained using quantum chemical calculations is found to be smaller than that obtained from solution NMR measurements, whereas the trends of the variations are found to be in close agreement. We believe that the results reported in this study will be useful in studying the structure and dynamics of membrane proteins and heme-containing proteins, and also membrane-bound protein-protein complexes such as cytochromes-b5-P450. PMID:23268659

  11. Multiple sources of carbonic anhydrase activity in pea thylakoids: soluble and membrane-bound forms.

    PubMed

    Rudenko, Natalia N; Ignatova, Lyudmila K; Ivanov, Boris N

    2007-01-01

    Carbonic anhydrase (CA) activity of pea thylakoids, thylakoid membranes enriched with photosystem I (PSI-membranes), or photosystem II (PSII-membranes) as well as both supernatant and pellet after precipitation of thylakoids treated with detergent Triton X-100 were studied. CA activity of thylakoids in the presence of varying concentrations of Triton X-100 had two maxima, at Triton/chlorophyll (triton/Chl) ratios of 0.3 and 1.0. CA activities of PSI-membranes and PSII-membranes had only one maximum each, at Triton/Chl ratio 0.3 or 1.0, respectively. Two CAs with characteristics of the membrane-bound proteins and one CA with characteristics of the soluble proteins were found in the medium after thylakoids were incubated with Triton. One of the first two CAs had mobility in PAAG after native electrophoresis the same as that of CA residing in PSI-membranes, and the other CA had mobility the same as the mobility of CA residing in PSII-membranes, but the latter was different from CA situated in PSII core-complex (Ignatova et al. 2006 Biochemistry (Moscow) 71:525-532). The properties of the "soluble" CA removed from thylakoids were different from the properties of the known soluble CAs of plant cell: apparent molecular mass was about 262 kD and it was three orders more sensitive to the specific CA inhibitor, ethoxyzolamide, than soluble stromal CA. The data are discussed as indicating the presence of, at least, four CAs in pea thylakoids. PMID:17347907

  12. Antigenic determinants of the membrane-bound hydrogenase in Alcaligenes eutrophus are exposed toward the periplasm.

    PubMed Central

    Eismann, K; Mlejnek, K; Zipprich, D; Hoppert, M; Gerberding, H; Mayer, F

    1995-01-01

    Electron microscopic immunogold labeling experiments were performed with ultrathin sections of plasmolyzed cells of Alcaligenes eutrophus and "whole-mount" samples of spheroplasts and protoplasts. They demonstrated that antigenic determinants of the membrane-bound hydrogenase are exposed, at the outside of the cytoplasmic membrane, to the periplasm. PMID:7592402

  13. AAA proteases in mitochondria: diverse functions of membrane-bound proteolytic machines.

    PubMed

    Tatsuta, Takashi; Langer, Thomas

    2009-11-01

    FtsH/AAA proteases comprise a distinct family of membrane-bound, ATP-dependent proteases present in eubacteria and eukaryotic cells, where they are confined to mitochondria and chloroplasts. Here, we will summarize versatile functions of AAA proteases within mitochondria, which ensure mitochondrial integrity and cell survival, acting both as quality control and processing enzymes. PMID:19781639

  14. On the chimerical nature of the membrane-bound ATPase from halobacterium saccharovorum

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.

    1991-01-01

    A series of experiments are described that were carried out with the goal of determining how the membrane-bound ATPase from H. saccharovorum is related to V- and F-type ATPases. They reflect three approaches: the use of inhibitors; structural studies; and immunological relatedness.

  15. Purification and characterization of membrane-bound semicarbazide-sensitive amine oxidase (SSAO) from bovine lung.

    PubMed Central

    Lizcano, J M; Tipton, K F; Unzeta, M

    1998-01-01

    Semicarbazide-sensitive amine oxidase (SSAO) has been purified from bovine lung microsomes in a form which is catalytically active and stable to storage. The enzyme, an integral membrane protein, was solubilized with Triton X-100 and purification was achieved, in the presence of detergent, by chromatography with Cibacron Blue 3GA-agarose, hydroxylapatite, Lens culinaris-agarose, Resource Q-FPLC and gel filtration on Superdex 200 HR-FPLC. This is the first reported procedure for the extensive purification of a membrane-bound SSAO. The purified enzyme had an apparent Mr of 400000 but exhibited microheterogeneity with SDS/PAGE and isoelectric focusing, probably as a result of its glycoprotein nature. It behaved as a tetramer with subunits with apparent Mr values of 100. Antibodies raised towards the purified enzyme cross-reacted with the enzymes from human lung and bovine plasma. Redox-cycling staining and reaction with carbonyl reagents were consistent with the presence of a quinone cofactor, possibly topa quinone. The enzyme was also shown to contain two mol of Cu/mol of enzyme and removal of half of this bound copper resulted essentially in complete inhibition of enzyme activity. In contrast to the reported behaviour of the SSAO enzymes from plasma, the bovine lung enzyme was relatively insensitive to inhibition by cyanide, copper-chelating agents and amiloride. The specificity of the bovine lung enzyme was also narrower than reported for soluble SSAO. It catalysed the oxidative deamination of benzylamine, methylamine, 2-phenylethylamine and histamine but had no significant activity towards dopamine, 5-hydroxytryptamine, tryptamine or tyramine. PMID:9512463

  16. Membrane-Bound TRAIL Supplements Natural Killer Cell Cytotoxicity Against Neuroblastoma Cells

    PubMed Central

    Sheard, Michael A.; Asgharzadeh, Shahab; Liu, Yin; Lin, Tsen-Yin; Wu, Hong-Wei; Ji, Lingyun; Groshen, Susan; Lee, Dean A.; Seeger, Robert C.

    2013-01-01

    Neuroblastoma cells have been reported to be resistant to death induced by soluble, recombinant forms of TRAIL (CD253/TNFSF10) due to low or absent expression of caspase-8 and/or TRAIL-receptor 2 (TRAIL-R2/DR5/CD262/TNFRSF10b). However, their sensitivity to membrane-bound TRAIL on natural killer (NK) cells is not known. Comparing microarray gene expression and response to NK cell-mediated cytotoxicity, we observed a correlation between TRAIL-R2 expression and the sensitivity of fourteen neuroblastoma cell lines to the cytotoxicity of NK cells activated with IL-2 plus IL-15. Even though most NK cytotoxicity was dependent upon perforin, the cytotoxicity was supplemented by TRAIL in fourteen of seventeen (82%) neuroblastoma cell lines as demonstrated using an anti-TRAIL neutralizing antibody. Similarly, a recently developed NK cell expansion system employing IL-2 plus lethally irradiated K562 feeder cells constitutively expressing membrane-bound IL-21 (K562 clone 9.mbIL21) resulted in activated NK cells derived from normal healthy donors and neuroblastoma patients that also utilized TRAIL to supplement cytotoxicity. Exogenous IFNγ up-regulated expression of caspase-8 in three of four neuroblastoma cell lines and increased the contribution of TRAIL to NK cytotoxicity against two of the three lines; however, relatively little inhibition of cytotoxicity was observed when activated NK cells were treated with an anti-IFNγ neutralizing antibody. Constraining the binding of anti-TRAIL neutralizing antibody to membrane-bound TRAIL but not soluble TRAIL indicated that membrane-bound TRAIL alone was responsible for essentially all of the supplemental cytotoxicity. Together, these findings support a role for membrane-bound TRAIL in the cytotoxicity of NK cells against neuroblastoma cells. PMID:23719242

  17. Reversible dissociation of flavin mononucleotide from the mammalian membrane-bound NADH:ubiquinone oxidoreductase (complex I)

    PubMed Central

    Gostimskaya, Irina S.; Grivennikova, Vera G.; Cecchini, Gary; Vinogradov, Andrei D.

    2008-01-01

    Conditions for the reversible dissociation of flavin mononucleotide (FMN) from the membrane-bound mitochondrial NADH:ubiquinone oxidoreductase (complex I) are described. The catalytic activities of the enzyme, i.e. rotenone-insensitive NADH:hexaammineruthenium III reductase and rotenone-sensitive NADH:quinone reductase decline when bovine heart submitochondrial particles are incubated with NADH in the presence of rotenone or cyanide at alkaline pH. FMN protects and fully restores the NADH-induced inactivation whereas riboflavin and flavin adenine dinucleotide do not. The data show that the reduction of complex I significantly weakens the binding of FMN to protein thus resulting in its dissociation when the concentration of holoenzyme is comparable with Kd (~10−8 M at pH 10.0). PMID:18037377

  18. A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data*

    PubMed Central

    Ahuja, Shivani; Jahr, Nicole; Im, Sang-Choul; Vivekanandan, Subramanian; Popovych, Nataliya; Le Clair, Stéphanie V.; Huang, Rui; Soong, Ronald; Xu, Jiadi; Yamamoto, Kazutoshi; Nanga, Ravi P.; Bridges, Angela; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2013-01-01

    Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb5 using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb5 and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb5. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes. PMID:23709268

  19. Fatty acyl donor selectivity in membrane bound O-acyltransferases and communal cell fate decision-making

    PubMed Central

    Tuladhar, Rubina; Lum, Lawrence

    2015-01-01

    The post-translational modification of proteins with lipid moieties confers spatial and temporal control of protein function by restricting their subcellular distribution or movement in the extracellular milieu. Yet, little is known about the significance of lipid selectivity to the activity of proteins targeted for such modifications. Membrane bound O-acyl transferases (MBOATs) are a superfamily of multipass enzymes that transfer fatty acids on to lipid or protein substrates. Three MBOATs constitute a subfamily with secreted signalling molecules for substrates, the Wnt, Hedgehog (Hh) and Ghrelin proteins. Given their important roles in adult tissue homoeostasis, all three molecules and their respective associated acyltransferases provide a framework for interrogating the role of extracellular acylation events in cell-to-cell communication. Here, we discuss how the preference for a fatty acyl donor in the Wnt acyltransferase porcupine (Porcn) and possibly in other protein lipidation enzymes may provide a means for coupling metabolic health at the single cell level to communal cell fate decision-making in complex multicellular organisms. PMID:25849923

  20. Fatty acyl donor selectivity in membrane bound O-acyltransferases and communal cell fate decision-making.

    PubMed

    Tuladhar, Rubina; Lum, Lawrence

    2015-04-01

    The post-translational modification of proteins with lipid moieties confers spatial and temporal control of protein function by restricting their subcellular distribution or movement in the extracellular milieu. Yet, little is known about the significance of lipid selectivity to the activity of proteins targeted for such modifications. Membrane bound O-acyl transferases (MBOATs) are a superfamily of multipass enzymes that transfer fatty acids on to lipid or protein substrates. Three MBOATs constitute a subfamily with secreted signalling molecules for substrates, the Wnt, Hedgehog (Hh) and Ghrelin proteins. Given their important roles in adult tissue homoeostasis, all three molecules and their respective associated acyltransferases provide a framework for interrogating the role of extracellular acylation events in cell-to-cell communication. Here, we discuss how the preference for a fatty acyl donor in the Wnt acyltransferase porcupine (Porcn) and possibly in other protein lipidation enzymes may provide a means for coupling metabolic health at the single cell level to communal cell fate decision-making in complex multicellular organisms. PMID:25849923

  1. Optimization of solubilization and purification procedures for the hydroxylase component of membrane-bound methane monooxygenase from Methylococcus capsulatus strain M.

    PubMed

    Vasil'ev, V I; Tikhonova, T V; Gvozdev, R I; Tukhvatullin, I A; Popov, V O

    2006-12-01

    The hydroxylase component of membrane-bound (particulate) methane monooxygenase (pMMO) from Methylococcus capsulatus strain M was isolated and purified to homogeneity. The pMMO molecule comprises three subunits of molecular masses 47, 26, and 23 kD and contains three copper atoms and one iron atom. In solution the protein exists as a stable oligomer of 660 kD with possible subunit composition (alpha beta gamma)6. Mass spectroscopy shows high homology of the purified protein with methane monooxygenase from Methylococcus capsulatus strain Bath. Pilot screening of crystallization conditions has been carried out. PMID:17223785

  2. Lyotropic Liquid Crystalline Cubic Phases as Versatile Host Matrices for Membrane-Bound Enzymes.

    PubMed

    Sun, Wenjie; Vallooran, Jijo J; Fong, Wye-Khay; Mezzenga, Raffaele

    2016-04-21

    Lyotropic liquid crystalline cubic mesophases can function as host matrices for enzymes because of their biomimetic structural characteristics, optical transparency, and capability to coexist with water. This study demonstrates that the in meso immobilized membrane-bound enzyme d-fructose dehydrogenase (FDH) preserves its full activity, follows ideal Michaelis-Menten kinetics, and shows improved stability compared to its behavior in solution. Even after 5 days, the immobilized FDH retained its full activity in meso, whereas a model hydrophilic enzyme, horseradish peroxidase, maintained only 21% of its original activity. We reason that the lipidic bilayers in the three-dimensional structures of cubic mesophases provide an ideal environment for the reconstitution of a membrane-bound enzyme. The preserved activity, long-term stability, and reusability demonstrate that these hybrid nanomaterials are ideal matrices for biosensing and biocatalytic fuel cell applications. PMID:27050734

  3. Sensing Size through Clustering in Non-Equilibrium Membranes and the Control of Membrane-Bound Enzymatic Reactions.

    PubMed

    Vagne, Quentin; Turner, Matthew S; Sens, Pierre

    2015-01-01

    The formation of dynamical clusters of proteins is ubiquitous in cellular membranes and is in part regulated by the recycling of membrane components. We show, using stochastic simulations and analytic modeling, that the out-of-equilibrium cluster size distribution of membrane components undergoing continuous recycling is strongly influenced by lateral confinement. This result has significant implications for the clustering of plasma membrane proteins whose mobility is hindered by cytoskeletal "corrals" and for protein clustering in cellular organelles of limited size that generically support material fluxes. We show how the confinement size can be sensed through its effect on the size distribution of clusters of membrane heterogeneities and propose that this could be regulated to control the efficiency of membrane-bound reactions. To illustrate this, we study a chain of enzymatic reactions sensitive to membrane protein clustering. The reaction efficiency is found to be a non-monotonic function of the system size, and can be optimal for sizes comparable to those of cellular organelles. PMID:26656912

  4. Sensing Size through Clustering in Non-Equilibrium Membranes and the Control of Membrane-Bound Enzymatic Reactions

    PubMed Central

    Vagne, Quentin; Turner, Matthew S.; Sens, Pierre

    2015-01-01

    The formation of dynamical clusters of proteins is ubiquitous in cellular membranes and is in part regulated by the recycling of membrane components. We show, using stochastic simulations and analytic modeling, that the out-of-equilibrium cluster size distribution of membrane components undergoing continuous recycling is strongly influenced by lateral confinement. This result has significant implications for the clustering of plasma membrane proteins whose mobility is hindered by cytoskeletal “corrals” and for protein clustering in cellular organelles of limited size that generically support material fluxes. We show how the confinement size can be sensed through its effect on the size distribution of clusters of membrane heterogeneities and propose that this could be regulated to control the efficiency of membrane-bound reactions. To illustrate this, we study a chain of enzymatic reactions sensitive to membrane protein clustering. The reaction efficiency is found to be a non-monotonic function of the system size, and can be optimal for sizes comparable to those of cellular organelles. PMID:26656912

  5. Rubredoxin-related Maturation Factor Guarantees Metal Cofactor Integrity during Aerobic Biosynthesis of Membrane-bound [NiFe] Hydrogenase*

    PubMed Central

    Fritsch, Johannes; Siebert, Elisabeth; Priebe, Jacqueline; Zebger, Ingo; Lendzian, Friedhelm; Teutloff, Christian; Friedrich, Bärbel; Lenz, Oliver

    2014-01-01

    The membrane-bound [NiFe] hydrogenase (MBH) supports growth of Ralstonia eutropha H16 with H2 as the sole energy source. The enzyme undergoes a complex biosynthesis process that proceeds during cell growth even at ambient O2 levels and involves 14 specific maturation proteins. One of these is a rubredoxin-like protein, which is essential for biosynthesis of active MBH at high oxygen concentrations but dispensable under microaerobic growth conditions. To obtain insights into the function of HoxR, we investigated the MBH protein purified from the cytoplasmic membrane of hoxR mutant cells. Compared with wild-type MBH, the mutant enzyme displayed severely decreased hydrogenase activity. Electron paramagnetic resonance and infrared spectroscopic analyses revealed features resembling those of O2-sensitive [NiFe] hydrogenases and/or oxidatively damaged protein. The catalytic center resided partially in an inactive Niu-A-like state, and the electron transfer chain consisting of three different Fe-S clusters showed marked alterations compared with wild-type enzyme. Purification of HoxR protein from its original host, R. eutropha, revealed only low protein amounts. Therefore, recombinant HoxR protein was isolated from Escherichia coli. Unlike common rubredoxins, the HoxR protein was colorless, rather unstable, and essentially metal-free. Conversion of the atypical iron-binding motif into a canonical one through genetic engineering led to a stable reddish rubredoxin. Remarkably, the modified HoxR protein did not support MBH-dependent growth at high O2. Analysis of MBH-associated protein complexes points toward a specific interaction of HoxR with the Fe-S cluster-bearing small subunit. This supports the previously made notion that HoxR avoids oxidative damage of the metal centers of the MBH, in particular the unprecedented Cys6[4Fe-3S] cluster. PMID:24448806

  6. Cellular chaperones and folding enzymes are vital contributors to membrane bound replication and movement complexes during plant RNA virus infection

    PubMed Central

    Verchot, Jeanmarie

    2012-01-01

    Cellular chaperones and folding enzymes play central roles in the formation of positive-strand and negative-strand RNA virus infection. This article examines the key cellular chaperones and discusses evidence that these factors are diverted from their cellular functions to play alternative roles in virus infection. For most chaperones discussed, their primary role in the cell is to ensure protein quality control. They are system components that drive substrate protein folding, complex assembly or disaggregation. Their activities often depend upon co-chaperones and ATP hydrolysis. During plant virus infection, Hsp70 and Hsp90 proteins play central roles in the formation of membrane-bound replication complexes for certain members of the tombusvirus, tobamovirus, potyvirus, dianthovirus, potexvirus, and carmovirus genus. There are several co-chaperones, including Yjd1, RME-8, and Hsp40 that associate with the bromovirus replication complex, pomovirus TGB2, and tospovirus Nsm movement proteins. There are also examples of plant viruses that rely on chaperone systems in the endoplasmic reticulum (ER) to support cell-to-cell movement. TMV relies on calreticulin to promote virus intercellular transport. Calreticulin also resides in the plasmodesmata and plays a role in calcium sequestration as well as glycoprotein folding. The pomovirus TGB2 interacts with RME-8 in the endosome. The potexvirus TGB3 protein stimulates expression of ER resident chaperones via the bZIP60 transcription factor. Up-regulating factors involved in protein folding may be essential to handling the load of viral proteins translated along the ER. In addition, TGB3 stimulates SKP1 which is a co-factor in proteasomal degradation of cellular proteins. Such chaperones and co-factors are potential targets for antiviral defense. PMID:23230447

  7. The Membrane-Bound Form of IL-17A Promotes the Growth and Tumorigenicity of Colon Cancer Cells

    PubMed Central

    Van Anh, Do Thi; Park, Sang Min; Lee, Hayyoung; Kim, Young Sang

    2016-01-01

    Interleukin-17A is a member of the IL-17 family, and is known as CTLA8 in the mouse. It is produced by T lymphocytes and NK cells and has proinflammatory roles, inducing cytokine and chemokine production. However, its role in tumor biology remains controversial. We investigated the effects of locally produced IL-17A by transferring the gene encoding it into CT26 colon cancer cells, either in a secretory or a membrane-bound form. Expression of the membrane-bound form on CT26 cells dramatically enhanced their proliferation in vitro. The enhanced growth was shown to be due to an increased rate of cell cycle progression: after synchronizing cells by adding and withdrawing colcemid, the rate of cell cycle progression in the cells expressing the membrane-bound form of IL-17A was much faster than that of the control cells. Both secretory and membrane-bound IL-17A induced the expression of Sca-1 in the cancer cells. When tumor clones were grafted into syngeneic BALB/c mice, the tumor clones expressing the membrane-bound form IL-17A grew rapidly; those expressing the secretory form also grew faster than the wild type CT26 cells, but slower than the clones expressing the membrane-bound form. These results indicate that IL-17A promotes tumorigenicity by enhancing cell cycle progression. This finding should be considered in treating tumors and immune-related diseases. PMID:27378226

  8. Hydrogen Production by a Hyperthermophilic Membrane-Bound Hydrogenase in Soluble Nanolipoprotein Particles

    SciTech Connect

    Baker, S E; Hopkins, R C; Blanchette, C; Walsworth, V; Sumbad, R; Fischer, N; Kuhn, E; Coleman, M; Chromy, B; Letant, S; Hoeprich, P; Adams, M W; Henderson, P T

    2008-10-22

    Hydrogenases constitute a promising class of enzymes for ex vivo hydrogen production. Implementation of such applications is currently hindered by oxygen sensitivity and, in the case of membrane-bound hydrogenases (MBH), poor water solubility. Nanolipoprotein particles (NLPs), formed from apolipoproteins and phospholipids, offer a novel means to incorporate MBH into in a well-defined water-soluble matrix that maintains the enzymatic activity and is amenable to incorporation into more complex architectures. We report the synthesis, hydrogen-evolving activity and physical characterization of the first MBH-NLP assembly. This may ultimately lead to the development of biomimetic hydrogen production devices.

  9. Molecular characterization of structural genes coding for a membrane bound hydrogenase in Methylococcus capsulatus (Bath).

    PubMed

    Csáki, R; Hanczár, T; Bodrossy, L; Murrell, J C; Kovács, K L

    2001-12-18

    The first gene cluster encoding for a membrane bound [NiFe] hydrogenase from a methanotroph, Methylococcus capsulatus (Bath), was cloned and sequenced. The cluster consisted of the structural genes hupS and hupL and accessory genes hupE, hupC and hupD. A DeltahupSL deletion mutant of Mc. capsulatus was constructed by marker exchange mutagenesis. Membrane associated hydrogenase activity disappeared. The membrane associated hydrogenase appeared to have a hydrogen uptake function in vivo. PMID:11750803

  10. An organelle-free assay for pea chloroplast Mg-chelatase: Resolution of the activity into soluble and membrane bound fractions

    SciTech Connect

    Walker, C.J.; Weinstein, J.D. )

    1991-05-01

    Mg-chelatase, which catalyzes the insertion of magnesium into protoporphyrin, lies at the branchpoint of heme and chlorophyll biosynthesis in chloroplasts. Since magnesium chelation is the first step unique to chlorophyll synthesis, one would expect this step to be highly regulated. However, to date little is known about the enzymology or regulation of Mg-chelatase due mostly to an inability to assay it's activity outside of the intact plastid. Here the authors report the first truly in vitro i.e. organelle-free, assay for Mg-chelatase. Mg-chelatase activity in intact pea chloroplasts which is 3 to 4 fold higher than in cucumber chloroplasts, survived chloroplast lysis and could be fractionated, by centrifugation, into supernatant and pellet components. Both of these fractions were required to reconstitute Mg-chelatase activity and both were inactivated by boiling; indicating that the enzyme is composed of soluble and membrane bound protein(s). The specific activity of the reconstituted system was typically 1 nmol Mg-Deuteroporphyrin/h/mg protein and activity was linear for at least 60 min under our assay conditions. ATP and magnesium were required for Mg-chelatase activity. The soluble component could be fractionated with ammonium sulfate. The product of the reaction was confirmed fluorometrically as the magnesium chelate of the porphyrin substrate. Crude separation of chloroplast membranes into thylakoids and envelopes, suggested that the membrane-bound component of Mg-chelatase is probably located in the envelope.

  11. Reduced Levels of Membrane-Bound Alkaline Phosphatase Are Common to Lepidopteran Strains Resistant to Cry Toxins from Bacillus thuringiensis

    PubMed Central

    Jurat-Fuentes, Juan Luis; Karumbaiah, Lohitash; Jakka, Siva Rama Krishna; Ning, Changming; Liu, Chenxi; Wu, Kongming; Jackson, Jerreme; Gould, Fred; Blanco, Carlos; Portilla, Maribel; Perera, Omaththage; Adang, Michael

    2011-01-01

    Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Identification of biomarkers would assist in the development of sensitive DNA-based methods to monitor evolution of resistance to Bt toxins in natural populations. We report on the proteomic and genomic detection of reduced levels of midgut membrane-bound alkaline phosphatase (mALP) as a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to susceptible larvae. Reduced levels of H. virescens mALP protein (HvmALP) were detected by two dimensional differential in-gel electrophoresis (2D-DIGE) analysis in Cry-resistant compared to susceptible larvae, further supported by alkaline phosphatase activity assays and Western blotting. Through quantitative real-time polymerase chain reaction (qRT-PCR) we demonstrate that the reduction in HvmALP protein levels in resistant larvae are the result of reduced transcript amounts. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived strains of S. frugiperda resistant to Cry1Fa. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest that reduced mALP expression should be targeted for development of effective biomarkers for resistance to Cry toxins in lepidopteran pests. PMID:21390253

  12. Membrane-bound human SCF/KL promotes in vivo human hematopoietic engraftment and myeloid differentiation

    PubMed Central

    Takagi, Shinsuke; Saito, Yoriko; Hijikata, Atsushi; Tanaka, Satoshi; Watanabe, Takashi; Hasegawa, Takanori; Mochizuki, Shinobu; Kunisawa, Jun; Kiyono, Hiroshi; Koseki, Haruhiko; Ohara, Osamu; Saito, Takashi; Taniguchi, Shuichi; Shultz, Leonard D.

    2012-01-01

    In recent years, advances in the humanized mouse system have led to significantly increased levels of human hematopoietic stem cell (HSC) engraftment. The remaining limitations in human HSC engraftment and function include lymphoid-skewed differentiation and inefficient myeloid development in the recipients. Limited human HSC function may partially be attributed to the inability of the host mouse microenvironment to provide sufficient support to human hematopoiesis. To address this problem, we created membrane-bound human stem cell factor (SCF)/KIT ligand (KL)–expressing NOD/SCID/IL2rgKO (hSCF Tg NSG) mice. hSCF Tg NSG recipients of human HSCs showed higher levels of both human CD45+ cell engraftment and human CD45+CD33+ myeloid development compared with NSG recipients. Expression of hSCF/hKL accelerated the differentiation of the human granulocyte lineage cells in the recipient bone marrow. Human mast cells were identified in bone marrow, spleen, and gastrointestinal tissues of the hSCF Tg NSG recipients. This novel in vivo humanized mouse model demonstrates the essential role of membrane-bound hSCF in human myeloid development. Moreover, the hSCF Tg NSG humanized recipients may facilitate investigation of in vivo differentiation, migration, function, and pathology of human mast cells. PMID:22279057

  13. Membrane-bound globin X protects the cell from reactive oxygen species.

    PubMed

    Koch, Jonas; Burmester, Thorsten

    2016-01-01

    Globin X (GbX) is a member of the globin family that emerged early in the evolution of Metazoa. In vertebrates, GbX is restricted to lampreys, fish, amphibians and some reptiles, and is expressed in neurons. Unlike any other metazoan globin, GbX is N-terminally acylated and anchored in the cell membrane via myristoyl and palmitoyl groups, suggesting a unique function. Here, we compared the capacity of GbX to protect a mouse neuronal cell line from hypoxia and reactive oxygen species (ROS) with that of myoglobin. To evaluate the contribution of membrane-binding, we generated a mutated version of GbX without acyl groups. All three globins enhanced cell viability under hypoxia, with myoglobin having the most pronounced effect. GbX but not myoglobin protected the cells from hydrogen peroxide (H2O2)-induced stress. Membrane-bound GbX was significantly more efficient than its mutated, soluble form. Furthermore, myoglobin and mutated GbX increased production of ROS upon H2O2-treatment, while membrane-bound GbX did not. The results indicate that myoglobin enhances O2 supply while GbX protects the cell membrane from ROS-stress. The ancient origin of GbX suggests that ROS-protection reflects the function of the early globins before they acquired a respiratory role. PMID:26631962

  14. Arrhenius plot behavior of a. gamma. -radiation-releasable, membrane-bound exonuclease

    SciTech Connect

    Mitchel, R.E.J.

    1981-11-01

    The activation energy of a membrane-bound exonuclease in Micrococcus radiodurans has been measured and the effect of ionizing radiation damage in this sytem explore. The Arrhenius plot for the native bound enzyme was found to be biphasic and the calculated activation energies and transition temperature for the enzymatic reaction were not changed when the enzyme was: (1) solubilized from the membrane with its covalently bound lipid anchor attached, (2) released from the membrane by ionizing radiation, which cleaves off the covalently attached lipid and converts the enzyme from a dimer to a monomer, (3) attached to the membrane after exposure to ionizing radiation under oxic or anoxic conditions, and (4) attached to the membrane in the presence of 10 mM CHCl/sub 3/. Since other membrane-bound enzymes have been shown to be sensitive to membrane perturbations while this one was not, the results suggest that various perturbants, including ionizing radiation, may have differential effects on such enzymes.

  15. The Tumor Necrosis Factor Receptor Stalk Regions Define Responsiveness to Soluble versus Membrane-Bound Ligand

    PubMed Central

    Richter, Christine; Messerschmidt, Sylvia; Holeiter, Gerlinde; Tepperink, Jessica; Osswald, Sylvia; Zappe, Andrea; Branschädel, Marcus; Boschert, Verena; Mann, Derek A.; Scheurich, Peter

    2012-01-01

    The family of tumor necrosis factor receptors (TNFRs) and their ligands form a regulatory signaling network that controls immune responses. Various members of this receptor family respond differently to the soluble and membrane-bound forms of their respective ligands. However, the determining factors and underlying molecular mechanisms of this diversity are not yet understood. Using an established system of chimeric TNFRs and novel ligand variants mimicking the bioactivity of membrane-bound TNF (mTNF), we demonstrate that the membrane-proximal extracellular stalk regions of TNFR1 and TNFR2 are crucial in controlling responsiveness to soluble TNF (sTNF). We show that the stalk region of TNFR2, in contrast to the corresponding part of TNFR1, efficiently inhibits both the receptor's enrichment/clustering in particular cell membrane regions and ligand-independent homotypic receptor preassembly, thereby preventing sTNF-induced, but not mTNF-induced, signaling. Thus, the stalk regions of the two TNFRs not only have implications for additional TNFR family members, but also provide potential targets for therapeutic intervention. PMID:22547679

  16. Purification and characterization of the membrane-bound quinoprotein glucose dehydrogenase of Gluconacetobacter diazotrophicus PAL 5.

    PubMed

    Sará-Páez, Martin; Contreras-Zentella, Martha; Gómez-Manzo, Saúl; González-Valdez, Alejandra Abigail; Gasca-Licea, Rolando; Mendoza-Hernández, Guillermo; Escamilla, José Edgardo; Reyes-Vivas, Horacio

    2015-02-01

    Acetic acid bacteria oxidize a great number of substrates, such as alcohols and sugars, using different enzymes that are anchored to the membrane. In particular, Gluconacetobacter diazotrophicus is distinguished for its N2-fixing activity under high-aeration conditions. Ga. diazotrophicus is a true endophyte that also has membrane-bound enzymes to oxidize sugars and alcohols. Here we reported the purification and characterization of the membrane-bound glucose dehydrogenase (GDHm), an oxidoreductase of Ga. diazotrophicus. GDHm was solubilized and purified by chromatographic methods. Purified GDHm was monomeric, with a molecular mass of 86 kDa. We identified the prosthetic group as pyrroloquinoline quinone, whose redox state was reduced. GDHm showed an optimum pH of 7.2, and its isoelectric point was 6.0. This enzyme preferentially oxidized D-glucose, 2-deoxy-D-glucose, D-galactose and D-xylose; its affinity towards glucose was ten times greater than that of E. coli GDHm. Finally, Ga. diazotrophicus GDHm was capable of reducing quinones such as Q 1, Q 2, and decylubiquinone; this activity was entirely abolished in the presence of micromolar concentrations of the inhibitor, myxothiazol. Hence, our purification method yielded a highly purified GDHm whose molecular and kinetic parameters were determined. The possible implications of GDHm activity in the mechanism for reducing competitor microorganisms, as well as its participation in the respiratory system of Ga. diazotrophicus, are discussed. PMID:25576305

  17. Membrane-bound amylopullulanase is essential for starch metabolism of Sulfolobus acidocaldarius DSM639.

    PubMed

    Choi, Kyoung-Hwa; Cha, Jaeho

    2015-09-01

    Sulfolobus acidocaldarius DSM639 produced an acid-resistant membrane-bound amylopullulanase (Apu) during growth on starch as a sole carbon and energy source. The physiological role of Apu in starch metabolism was investigated by the growth and starch degradation pattern of apu disruption mutant as well as biochemical properties of recombinant Apu. The Δapu mutant lost the ability to grow in minimal medium in the presence of starch, and the amylolytic activity observed in the membrane fraction of the wild-type strain was not detected in the Δapu mutant when the cells were grown in YT medium. The purified membrane-bound Apu initially hydrolyzed starch, amylopectin, and pullulan into various sizes of maltooligosaccharides, and then produced glucose, maltose, and maltotriose in the end, indicating Apu is a typical endo-acting glycoside hydrolase family 57 (GH57) amylopullulanase. The maltose and maltotriose observed in the culture medium during the exponential and stationary phase growth indicates that Apu is the essential enzyme to initially hydrolyze the starch into small maltooligosaccharides to be transported into the cell. PMID:26104674

  18. Chelation of Membrane-Bound Cations by Extracellular DNA Activates the Type VI Secretion System in Pseudomonas aeruginosa.

    PubMed

    Wilton, Mike; Wong, Megan J Q; Tang, Le; Liang, Xiaoye; Moore, Richard; Parkins, Michael D; Lewenza, Shawn; Dong, Tao G

    2016-08-01

    Pseudomonas aeruginosa employs its type VI secretion system (T6SS) as a highly effective and tightly regulated weapon to deliver toxic molecules to target cells. T6SS-secreted proteins of P. aeruginosa can be detected in the sputum of cystic fibrosis (CF) patients, who typically present a chronic and polymicrobial lung infection. However, the mechanism of T6SS activation in the CF lung is not fully understood. Here we demonstrate that extracellular DNA (eDNA), abundant within the CF airways, stimulates the dynamics of the H1-T6SS cluster apparatus in Pseudomonas aeruginosa PAO1. Addition of Mg(2+) or DNase with eDNA abolished such activation, while treatment with EDTA mimicked the eDNA effect, suggesting that the eDNA-mediated effect is due to chelation of outer membrane-bound cations. DNA-activated H1-T6SS enables P. aeruginosa to nonselectively attack neighboring species regardless of whether or not it was provoked. Because of the importance of the T6SS in interspecies interactions and the prevalence of eDNA in the environments that P. aeruginosa inhabits, our report reveals an important adaptation strategy that likely contributes to the competitive fitness of P. aeruginosa in polymicrobial communities. PMID:27271742

  19. The rice thylakoid membrane-bound ascorbate peroxidase OsAPX8 functions in tolerance to bacterial blight

    PubMed Central

    Jiang, Guanghuai; Yin, Dedong; Zhao, Jiying; Chen, Honglin; Guo, Lequn; Zhu, Lihuang; Zhai, Wenxue

    2016-01-01

    Thylakoid membrane-bound ascorbate peroxidase (tAPX) is a major H2O2-scavenging enzyme. To clarify its functions in tolerance to rice bacterial blight, we produced rice lines overexpressing and suppressing tAPX (OsAPX8). The overexpressing lines exhibited increased tolerance to bacterial pathogen. The RNA interference (RNAi) lines were considerably more sensitive than the control plant. Further analysis of the H2O2 content in these transgenic plants indicated that the H2O2 accumulation of OsAPX8-overexpressing plants was considerably less than that of wild-type and RNAi plants upon challenge with bacterial pathogen. Interestingly, H2O2 was the most important factor for the serious leaf dehydration and withering of rice without major resistance genes and was not the cause of hypersensitivity. It addition, wall tightening or loosening can occur according to the level of H2O2. In addition, OsAPX8 interacted with the susceptibility protein Os8N3/Xa13, and their binding repressed the reaction of OsAPX8 in tolerance to bacterial blight. PMID:27185545

  20. An investigation into membrane bound redox carriers involved in energy transduction mechanism in Brevibacterium linens DSM 20158 with unsequenced genome.

    PubMed

    Shabbiri, Khadija; Botting, Catherine H; Adnan, Ahmad; Fuszard, Matthew; Naseem, Shahid; Ahmed, Safeer; Shujaat, Shahida; Syed, Quratulain; Ahmad, Waqar

    2014-04-01

    Brevibacterium linens (B. linens) DSM 20158 with an unsequenced genome can be used as a non-pathogenic model to study features it has in common with other unsequenced pathogens of the same genus on the basis of comparative proteome analysis. The most efficient way to kill a pathogen is to target its energy transduction mechanism. In the present study, we have identified the redox protein complexes involved in the electron transport chain of B. linens DSM 20158 from their clear homology with the shot-gun genome sequenced strain BL2 of B. linens by using the SDS-Polyacrylamide gel electrophoresis coupled with nano LC-MS/MS mass spectrometry. B. linens is found to have a branched electron transport chain (Respiratory chain), in which electrons can enter the respiratory chain either at NADH (Complex I) or at Complex II level or at the cytochrome level. Moreover, we are able to isolate, purify, and characterize the membrane bound Complex II (succinate dehydrogenase), Complex III (menaquinone cytochrome c reductase cytochrome c subunit, Complex IV (cytochrome c oxidase), and Complex V (ATP synthase) of B. linens strain DSM 20158. PMID:24573306

  1. Bacillus thuringiensis subsp. israelensis Cyt1Aa synergizes Cry11Aa toxin by functioning as a membrane-bound receptor.

    PubMed

    Pérez, Claudia; Fernandez, Luisa E; Sun, Jianguang; Folch, Jorge Luis; Gill, Sarjeet S; Soberón, Mario; Bravo, Alejandra

    2005-12-20

    Bacillus thuringiensis subsp. israelensis produces crystal proteins, Cry (4Aa, 4Ba, 10Aa, and 11Aa) and Cyt (1Aa and 2Ba) proteins, toxic to mosquito vectors of human diseases. Cyt1Aa overcomes insect resistance to Cry11Aa and Cry4 toxins and synergizes the toxicity of these toxins. However, the molecular mechanism of synergism remains unsolved. Here, we provide evidence that Cyt1Aa functions as a receptor of Cry11Aa. Sequential-binding analysis of Cyt1Aa and Cry11Aa revealed that Cyt1Aa binding to Aedes aegypti brush border membrane vesicles enhanced the binding of biotinylated-Cry11Aa. The Cyt1Aa- and Cry11Aa-binding epitopes were mapped by means of the yeast two-hybrid system, peptide arrays, and heterologous competition assays with synthetic peptides. Two exposed regions in Cyt1Aa, loop beta6-alphaE and part of beta7, bind Cry11Aa. On the other side, Cry11Aa binds Cyt1Aa proteins by means of domain II-loop alpha8 and beta-4, which are also involved in midgut receptor interaction. Characterization of single-point mutations in Cry11Aa and Cyt1Aa revealed key Cry11Aa (S259 and E266) and Cyt1Aa (K198, E204 and K225) residues involved in the interaction of both proteins and in synergism. Additionally, a Cyt1Aa loop beta6-alphaE mutant (K198A) with enhanced synergism to Cry11Aa was isolated. Data provided here strongly indicates that Cyt1Aa synergizes or suppresses resistance to Cry11Aa toxin by functioning as a membrane-bound receptor. Bacillus thuringiensis subsp. israelensis is a highly effective pathogenic bacterium because it produces a toxin and also its functional receptor, promoting toxin binding to the target membrane and causing toxicity. PMID:16339907

  2. Rapid effects of aldosterone in primary cultures of cardiomyocytes - do they suggest the existence of a membrane-bound receptor?

    PubMed

    Araujo, Carolina Morais; Hermidorff, Milla Marques; Amancio, Gabriela de Cassia Sousa; Lemos, Denise da Silveira; Silva, Marcelo Estáquio; de Assis, Leonardo Vinícius Monteiro; Isoldi, Mauro César

    2016-10-01

    Aldosterone acts on its target tissue through a classical mechanism or through the rapid pathway through a putative membrane-bound receptor. Our goal here was to better understand the molecular and biochemical rapid mechanisms responsible for aldosterone-induced cardiomyocyte hypertrophy. We have evaluated the hypertrophic process through the levels of ANP, which was confirmed by the analysis of the superficial area of cardiomyocytes. Aldosterone increased the levels of ANP and the cellular area of the cardiomyocytes; spironolactone reduced the aldosterone-increased ANP level and cellular area of cardiomyocytes. Aldosterone or spironolactone alone did not increase the level of cyclic 3',5'-adenosine monophosphate (cAMP), but aldosterone plus spironolactone led to increased cAMP level; the treatment with aldosterone + spironolactone + BAPTA-AM reduced the levels of cAMP. These data suggest that aldosterone-induced cAMP increase is independent of mineralocorticoid receptor (MR) and dependent on Ca(2+). Next, we have evaluated the role of A-kinase anchor proteins (AKAP) in the aldosterone-induced hypertrophic response. We have found that St-Ht31 (AKAP inhibitor) reduced the increased level of ANP which was induced by aldosterone; in addition, we have found an increase on protein kinase C (PKC) and extracellular signal-regulated kinase 5 (ERK5) activity when cells were treated with aldosterone alone, spironolactone alone and with a combination of both. Our data suggest that PKC could be responsible for ERK5 aldosterone-induced phosphorylation. Our study suggests that the aldosterone through its rapid effects promotes a hypertrophic response in cardiomyocytes that is controlled by an AKAP, being dependent on ERK5 and PKC, but not on cAMP/cAMP-dependent protein kinase signaling pathways. Lastly, we provide evidence that the targeting of AKAPs could be relevant in patients with aldosterone-induced cardiac hypertrophy and heart failure. PMID:27305962

  3. Genome-Based Discovery of a Novel Membrane-Bound 1,6-Dihydroxyphenazine Prenyltransferase from a Marine Actinomycete

    PubMed Central

    Zeyhle, Philipp; Bauer, Judith S.; Kalinowski, Jörn; Shin-ya, Kazuo; Gross, Harald; Heide, Lutz

    2014-01-01

    Recently, novel prenylated derivatives of 1,6-dihydroxyphenazine have been isolated from the marine sponge-associated Streptomyces sp. SpC080624SC-11. Genome sequencing of this strain now revealed a gene cluster containing all genes necessary for the synthesis of the phenazine and the isoprenoid moieties. Unexpectedly, however, the cluster did not contain a gene with similarity to previously investigated phenazine prenyltransferases, but instead a gene with modest similarity to the membrane-bound prenyltransferases of ubiquinone and menaquinone biosynthesis. Expression of this gene in E. coli and isolation of the membrane fraction proved that the encoded enzyme, Mpz10, catalyzes two successive prenylations of 1,6-dihydroxyphenazine. Mpz10 is the first example of a membrane-bound enzyme catalyzing the prenylation of a phenazine substrate, and one of few examples of membrane-bound enzymes involved in the prenylation of aromatic secondary metabolites in microorganisms. PMID:24892559

  4. The reaction pathway of membrane-bound rat liver mitochondrial monoamine oxidase

    PubMed Central

    Houslay, Miles D.; Tipton, Keith F.

    1973-01-01

    1. A preparation of a partly purified mitochondrial outer-membrane fraction suitable for kinetic investigations of monoamine oxidase is described. 2. An apparatus suitable for varying the O2 concentration in a spectrophotometer cuvette is described. 3. The reaction catalysed by the membrane-bound enzyme is shown to proceed by a double-displacement (Ping Pong) mechanism, and a formal mechanism is proposed. 4. KCN, NaN3, benzyl cyanide and 4-cyanophenol are shown to be reversible inhibitors of the enzyme. 5. The non-linear reciprocal plot obtained with impure preparations of benzylamine, which is typical of high substrate inhibition, is shown to be due to aldehyde contamination of the substrate. PMID:4778271

  5. Electrochemical insights into the mechanism of NiFe membrane-bound hydrogenases.

    PubMed

    Flanagan, Lindsey A; Parkin, Alison

    2016-02-15

    Hydrogenases are enzymes of great biotechnological relevance because they catalyse the interconversion of H2, water (protons) and electricity using non-precious metal catalytic active sites. Electrochemical studies into the reactivity of NiFe membrane-bound hydrogenases (MBH) have provided a particularly detailed insight into the reactivity and mechanism of this group of enzymes. Significantly, the control centre for enabling O2 tolerance has been revealed as the electron-transfer relay of FeS clusters, rather than the NiFe bimetallic active site. The present review paper will discuss how electrochemistry results have complemented those obtained from structural and spectroscopic studies, to present a complete picture of our current understanding of NiFe MBH. PMID:26862221

  6. The purification and subunit structure of a membrane-bound ATPase from the Archaebacterium Halobacterium saccharovorum

    NASA Technical Reports Server (NTRS)

    Hochstein, Lawrence I.; Kristjansson, Hordur; Altekar, Wijaya

    1987-01-01

    The procedure for the isolation and 70-fold purification of membrane-bound cold-sensitive ATPase from Halobacterium saccharovorum is described. Upon exposure to cold, the enzyme dissociates into two major subunits, I (87 kDa) and II (60 kDa), and two minor subunits, III (29 kDa) and IV (20 kDa). The stoichiometry of the enzyme is proposed to be I2.II2.III.IV; the molecular mass of such a complex would be 343 kDa, which is in good agreement with the value of 350 kDa obtained by gel filtration. The structure of the ATPase from H. saccharovorum makes it unlike any previously described ATPase.

  7. Electrochemical insights into the mechanism of NiFe membrane-bound hydrogenases

    PubMed Central

    Flanagan, Lindsey A.; Parkin, Alison

    2016-01-01

    Hydrogenases are enzymes of great biotechnological relevance because they catalyse the interconversion of H2, water (protons) and electricity using non-precious metal catalytic active sites. Electrochemical studies into the reactivity of NiFe membrane-bound hydrogenases (MBH) have provided a particularly detailed insight into the reactivity and mechanism of this group of enzymes. Significantly, the control centre for enabling O2 tolerance has been revealed as the electron-transfer relay of FeS clusters, rather than the NiFe bimetallic active site. The present review paper will discuss how electrochemistry results have complemented those obtained from structural and spectroscopic studies, to present a complete picture of our current understanding of NiFe MBH. PMID:26862221

  8. Methods for measuring Class I membrane-bound hyaluronan synthase activity.

    PubMed

    Weigel, Paul H; Padgett-McCue, Amy J; Baggenstoss, Bruce A

    2013-01-01

    Detecting and quantifying hyaluronan (HA) made by Class I HA synthase (HAS) and determining the level of activity of these membrane-bound enzymes is critical in studies to understand the normal biology of HA and how changes in HAS activity and HA levels or size are important in inflammatory and other diseases, tumorigenesis, and metastasis. Unlike the products made by the vast majority of glycosyltransferases, HA products are more complicated since they are made as a heterogeneous population of sizes spanning a broad mass range. Three radioactive and nonradioactive assay methods are described that can give the amount of HA made with or without information about the distribution of product sizes. PMID:23765666

  9. Top-Down Mass Spectrometry Analysis of Membrane-Bound Light-Harvesting Complex 2 from Rhodobacter sphaeroides.

    PubMed

    Lu, Yue; Zhang, Hao; Cui, Weidong; Saer, Rafael; Liu, Haijun; Gross, Michael L; Blankenship, Robert E

    2015-12-15

    We report a top-down proteomic analysis of the membrane-bound peripheral light-harvesting complex LH2 isolated from the purple photosynthetic bacterium Rhodobacter sphaeroides. The LH2 complex is coded for by the puc operon. The Rb. sphaeroides genome contains two puc operons, designated puc1BAC and puc2BA. Although previous work has shown consistently that the LH2 β polypeptide coded by the puc2B gene was assembled into LH2 complexes, there are contradictory reports as to whether the Puc2A polypeptides are incorporated into LH2 complexes. Furthermore, post-translational modifications of this protein offer the prospect that it could coordinate bacteriochlorophyll a (Bchl a) by a modified N-terminal residue. Here, we describe the components of the LH2 complex on the basis of electron-capture dissociation fragmentation to confirm the identity and sequence of the protein's subunits. We found that both gene products of the β polypeptides are expressed and assembled in the mature LH2 complex, but only the Puc1A-encoded polypeptide α is observed here. The methionine of the Puc2B-encoded polypeptide is missing, and a carboxyl group is attached to the threonine at the N-terminus. Surprisingly, one amino acid encoded as an isoleucine in both the puc2B gene and the mRNA is found as valine in the mature LH2 complex, suggesting an unexpected and unusual post-translational modification or a specific tRNA recoding of this one amino acid. PMID:26574182

  10. Nitrate reduction associated with respiration in Sinorhizobium meliloti 2011 is performed by a membrane-bound molybdoenzyme.

    PubMed

    Ferroni, Felix M; Rivas, María G; Rizzi, Alberto C; Lucca, María E; Perotti, Nora I; Brondino, Carlos D

    2011-10-01

    The purification and biochemical characterization of the respiratory membrane-bound nitrate reductase from Sinorhizobium meliloti 2011 (Sm NR) is reported together with the optimal conditions for cell growth and enzyme production. The best biomass yield was obtained under aerobic conditions in a fed-batch system using Luria-Bertani medium with glucose as carbon source. The highest level of Sm NR production was achieved using microaerobic conditions with the medium supplemented with both nitrate and nitrite. Sm NR is a mononuclear Mo-protein belonging to the DMSO reductase family isolated as a heterodimeric enzyme containing two subunits of 118 and 45 kDa. Protein characterization by mass spectrometry showed homology with respiratory nitrate reductases. UV-Vis spectra of as-isolated and dithionite reduced Sm NR showed characteristic absorption bands of iron-sulfur and heme centers. Kinetic studies indicate that Sm NR follows a Michaelis-Menten mechanism (K (m) = 97 ± 11 μM, V = 9.4 ± 0.5 μM min(-1), and k (cat) = 12.1 ± 0.6 s(-1)) and is inhibited by azide, chlorate, and cyanide with mixed inhibition patterns. Physiological and kinetic studies indicate that molybdenum is essential for NR activity and that replacement of this metal for tungsten inhibits the enzyme. Although no narGHI gene cluster has been annotated in the genome of rhizobia, the biochemical characterization indicates that Sm NR is a Mo-containing NR enzyme with molecular organization similar to NarGHI. PMID:21432624

  11. A membrane-bound synthetic receptor that promotes growth of a polymeric coating at the bilayer-water interface.

    PubMed

    Liu, Ying; Young, Michael C; Moshe, Orly; Cheng, Quan; Hooley, Richard J

    2012-07-27

    Primed for action: Atom-transfer radical polymerization (ATRP) can be promoted at a bilayer-water interface by anchoring initiator molecules (see scheme; red) in a membrane-bound synthetic receptor (yellow). The bilayer is formed on a calcinated nanofilm (gray) on a gold surface. PMID:22730162

  12. Overproduction of the membrane-bound [NiFe]-hydrogenase in Thermococcus kodakarensis and its effect on hydrogen production

    PubMed Central

    Kanai, Tamotsu; Simons, Jan-Robert; Tsukamoto, Ryohei; Nakajima, Akihito; Omori, Yoshiyuki; Matsuoka, Ryoji; Beppu, Haruki; Imanaka, Tadayuki; Atomi, Haruyuki

    2015-01-01

    The hyperthermophilic archaeon Thermococcus kodakarensis can utilize sugars or pyruvate for growth. In the absence of elemental sulfur, the electrons via oxidation of these substrates are accepted by protons, generating molecular hydrogen (H2). The hydrogenase responsible for this reaction is a membrane-bound [NiFe]-hydrogenase (Mbh). In this study, we have examined several possibilities to increase the protein levels of Mbh in T. kodakarensis by genetic engineering. Highest levels of intracellular Mbh levels were achieved when the promoter of the entire mbh operon (TK2080-TK2093) was exchanged to a strong constitutive promoter from the glutamate dehydrogenase gene (TK1431) (strain MHG1). When MHG1 was cultivated under continuous culture conditions using pyruvate-based medium, a nearly 25% higher specific hydrogen production rate (SHPR) of 35.3 mmol H2 g-dcw−1 h−1 was observed at a dilution rate of 0.31 h−1. We also combined mbh overexpression using an even stronger constitutive promoter from the cell surface glycoprotein gene (TK0895) with disruption of the genes encoding the cytosolic hydrogenase (Hyh) and an alanine aminotransferase (AlaAT), both of which are involved in hydrogen consumption (strain MAH1). At a dilution rate of 0.30 h−1, the SHPR was 36.2 mmol H2 g-dcw−1 h−1, corresponding to a 28% increase compared to that of the host T. kodakarensis strain. Increasing the dilution rate to 0.83 h−1 or 1.07 h−1 resulted in a SHPR of 120 mmol H2 g-dcw−1 h−1, which is one of the highest production rates observed in microbial fermentation. PMID:26379632

  13. A membrane-bound form of glutamate dehydrogenase possesses an ATP-dependent high-affinity microtubule-binding activity.

    PubMed Central

    Rajas, F; Rousset, B

    1993-01-01

    We previously identified a 50 kDa membrane protein which bound to in vitro assembled microtubules [Mithieux and Rousset (1989) J. Biol. Chem. 264, 4664-4668]. This protein exhibited the expected properties for mediating the ATP-dependent association of vesicles with microtubules [Mithieux, Audebet and Rousset (1988) Biochim. Biophys. Acta 969, 121-130]. The 50 kDa membrane protein (MP50), initially extracted in very low amount from isolated pig thyroid lysosomes/endosomes, has now been purified from membrane preparations of crude vesicle fractions from pig liver and brain. MP50 was isolated from detergent-solubilized membrane protein by affinity chromatography on immobilized ATP; 3-5 mg of MP50 was obtained from 100 g of liver tissue. Phase partitioning in Triton X-114 indicated that MP50 is a peripheral membrane protein. Radioiodinated liver MP50 bound to microtubules assembled in vitro. The binding was inhibited by ATP (Ki = 0.76 mM) and displaced by unlabelled liver or brain MP50. Equilibrium binding studies yielded KD values of 1.8 x 10(-7) M. By N-terminal amino acid sequence analysis, MP50 was identified as glutamate dehydrogenase (GDH), by comparison of V8 protease peptide maps of MP50 with purified liver GDH. Liver MP50 exhibited a low GDH activity; 4-5 units/mg compared with 18 and 34 units/mg for purified bovine and rat liver GDH respectively. Bovine and rat liver GDH yielded six spots from pI 5.7 to 7.2 when analysed by two-dimensional electrophoresis; in contrast, MP50 gave one main spot (corresponding to spot 2 of liver GDH) with a pI of approx. 6.5. Soluble liver GDH from commercial sources exhibited a very low or no microtubule-binding activity. In conclusion, we have found a membrane-bound form of GDH capable of specific and nucleotide-sensitive interaction with microtubules. Our data suggest that GDH isoproteins, the number of which has been undervalued up to now, could have cellular functions other than that of an enzyme. Images Figure 1 Figure 3

  14. Efficient photoinduced orthogonal energy and electron transfer reactions via phospholipid membrane-bound donors and acceptors

    SciTech Connect

    Clapp, P.J.; Armitage, B.; Roosa, P.; O'Brien, D.F. )

    1994-10-05

    A three component, liposome-bound photochemical molecular device (PMD) consisting of energy and electron transfer reactions is described. Bilayer membrane surface-associated dyes, 5,10,15,20-tetrakis[4-(trimethylammonio)-phenyl]-21H,2 3H-porphine tetra-p-tosylate salt and N,N[prime]-bis[(3-trimethylammonio)propyl]thiadicarbocya nine tribromide, are the energy donor and acceptor, respectively, in a blue light stimulated energy transfer reaction along the vesicle surface. The electronically excited cyanine is quenched by electron transfer from the phospholipid membrane bound triphenylbenzyl borate anion, which is located in the lipid bilayer interior. The PMD exhibits sequential reactions following electronic excitation with the novel feature that the steps proceed with orthogonal orientation: energy transfer occurs parallel to the membrane surface, and electron transfer occurs perpendicular to the surface. Photobleaching and fluorescence quenching experiments verify the transfer reactions, and Stern-Volmer analysis was used to estimate the reaction rate constants. At the highest concentrations examined of energy and electron acceptor ca. 60% of the photoexcited porphyrins were quenched by energy transfer to the cyanine. 56 refs., 6 figs., 3 tabs.

  15. Purification and structural analysis of membrane-bound polyphenol oxidase from Fuji apple.

    PubMed

    Liu, Fang; Zhao, Jin-Hong; Wen, Xin; Ni, Yuan-Ying

    2015-09-15

    Membrane-bound polyphenol oxidase (mPPO) in Fuji apple (Malus domestica Borkh. cv. Red Fuji) was purified and analyzed with a nanoelectrospray ionization mass spectrometer. The three-dimensional model and binding site of mPPO to 4-methyl catechol were also studied using molecular docking. mPPO was purified 54.41-fold using temperature-induced phase partitioning technique and ion exchange chromatography. mPPO had a molecular weight of 67.3kDa. Even though a significant level of homology was observed between mPPO and the soluble polyphenol oxidase in the copper binding sequence, there was another region, rich in histidine residues, which differed in 13 amino acids. The three-dimensional structure of mPPO consisted of six α-helices, two short β-strands, and ten random coils. The putative substrate-binding pocket contained six polar or charged amino acids, His191, His221, Trp224, Trp228, Phe227, and Val190. Trp224 and Trp228 formed hydrogen bonds with 4-methyl-catechol. PMID:25863612

  16. Development of a Membrane-Bound Random DNA Sequence Combinatorial Array Recognition Surface (CARS)

    PubMed Central

    Bruno, John G.

    2010-01-01

    A partially overlapping population of random sequence 60mer DNA molecules consisting of many concatamers of varied lengths was spatially separated in one and two dimensions by electrophoresis in polyacrylamide and transferred to nitrocellulose membranes. The spatially separated library serves as a potential sensor interface on which many different molecular recognition events or target analyte-binding patterns may emerge, thereby theoretically representing a “universal sensor” surface. The separated DNA library has been referred to as a DNA combinatorial array recognition surface or “CARS.” After UV baking and various fluorescence staining or fluorescent probe interactions, the one-dimensional (1-D) and 2-D membrane-bound CARS were digitally photographed and subjected to image analysis with National Institutes of Health Image-Java software. Image analysis demonstrated relatively consistent and more similar spatial fluorescence patterns within CARS analyte treatment groups but noteworthy pattern differences before and after analyte addition and between different analyte treatments. Taken together, these data suggest a potential role for CARS as a novel, inexpensive, self-assembling universal molecular recognition surface that could be coupled to sophisticated Bayesian or other pattern recognition algorithms to classify analytes or make specific identifications, much like the senses of smell or taste. PMID:20357981

  17. Diversity of Membrane-Bound Nitrate Reductase Genes in Geothermal Springs

    NASA Astrophysics Data System (ADS)

    Poret-Peterson, A. T.; Schwegel, R.; Elser, J. J.; Shock, E.; Anbar, A. D.

    2010-12-01

    Yellowstone National Park (YNP) harbors an array of hot springs with diverse geochemical properties encompassing gradients of pH (<1 to >9), temperature (ambient to boiling), oxygen levels, metal and nutrient concentrations. Such geothermal features provide ideal settings to study nitrogen (N) cycling in high temperature aquatic environments. Our current understanding of N cycle dynamics in hydrothermal systems comes mainly from the study of nitrogen fixation and nitrification. Indeed, research in these areas has extended the upper temperature limits for both processes to above 80°C and stimulated new thoughts on these processes at the cellular and organismal levels. Denitrification at elevated temperatures, on the other hand, has received com-paratively little attention. Here, we use functional gene markers to explore denitrification in YNP hydrothermal springs. During two consecutive summers, we collected sediment and microbial mat samples from various geothermal features for analysis of genes for denitrification and characterization of geochemical parameters (e.g., pH, temperature, relative abundance of trace metals, etc.). Genes encoding putative membrane-bound nitrate reductase (narG)were amplified from sediments and microbial mats of hot springs ranging in temperature from 50°C up to 92°C. Phylogenetic analysis of these genes show that they are most closely related to narG sequences from hyperthermophilic archaea.

  18. Purification and characterization of the membrane-bound ferrochelatase from Spirillum itersonii.

    PubMed Central

    Dailey, H A

    1977-01-01

    The membrane-bound enzyme ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) was purified from isolated membrane fragments of Spirillum itersonii approximately 490-fold. Purification was achieved by solubilization with chaotropic salts followed by ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, and gel filtration on Sephadex G-200. The purified enzyme has an apparent minimum molecular weight of approximately 50,000, as determined by gel filtration in the presence of 0.1% Brij 35 and 1 mM dithiothreitol but forms high-molecular-weight aggregates in the absence of detergent. Purified ferrochelatase is strongly stimulated in the presence of copper. The apparent Km for Fe2+ is 20 micrometer in the absence of copper and 9.5 micrometer in the presence of 20 micrometer CuCl2. The apparent Km for protoporphyrin is 50 micrometer, and it is unaltered by copper. Ferrochelatase has a single pH optimum of 7.50, and it is inhibited 50% by 20 micrometer heme. Certain divalent cations and sulfhydryl reagents also inhibit the enzyme. Images PMID:21163

  19. Overexpression of membrane-bound fas ligand (CD95L) exacerbates autoimmune disease and renal pathology in pristane-induced lupus.

    PubMed

    Bossaller, Lukas; Rathinam, Vijay A K; Bonegio, Ramon; Chiang, Ping-I; Busto, Patricia; Wespiser, Adam R; Caffrey, Daniel R; Li, Quan-Zhen; Mohan, Chandra; Fitzgerald, Katherine A; Latz, Eicke; Marshak-Rothstein, Ann

    2013-09-01

    Loss-of-function mutations in the Fas death receptor or its ligand result in a lymphoproliferative syndrome and exacerbate clinical disease in most lupus-prone strains of mice. One exception is mice injected with 2,6,10,14-tetramethylpentadecane (TMPD), a hydrocarbon oil commonly known as pristane, which induces systemic lupus erythematosus-like disease. Although Fas/Fas ligand (FasL) interactions have been strongly implicated in the activation-induced cell death of both lymphocytes and other APCs, FasL can also trigger the production of proinflammatory cytokines. FasL is a transmembrane protein with a matrix metalloproteinase cleavage site in the ectodomain. Matrix metalloproteinase cleavage inactivates membrane-bound FasL and releases a soluble form reported to have both antagonist and agonist activity. To better understand the impact of FasL cleavage on both the proapoptotic and proinflammatory activity of FasL, its cleavage site was deleted through targeted mutation to produce the deleted cleavage site (ΔCS) mouse line. ΔCS mice express higher levels of membrane-bound FasL than do wild-type mice and fail to release soluble FasL. To determine to what extent FasL promotes inflammation in lupus mice, TMPD-injected FasL-deficient and ΔCS BALB/c mice were compared with control TMPD-injected BALB/c mice. We found that FasL deficiency significantly reduced the early inflammatory exudate induced by TMPD injection. In contrast, ΔCS mice developed a markedly exacerbated disease profile associated with a higher frequency of splenic neutrophils and macrophages, a profound change in anti-nuclear Ab specificity, and markedly increased proteinuria and kidney pathology compared with controls. These results demonstrate that FasL promotes inflammation in TMPD-induced autoimmunity, and its cleavage limits FasL proinflammatory activity. PMID:23918976

  20. ATPaseTb2, a Unique Membrane-bound FoF1-ATPase Component, Is Essential in Bloodstream and Dyskinetoplastic Trypanosomes

    PubMed Central

    Šubrtová, Karolína; Panicucci, Brian; Zíková, Alena

    2015-01-01

    In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt) membrane potential (Δψm) is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential FoF1-ATPase. Intriguingly, this multiprotein complex contains several trypanosome-specific subunits of unknown function. Here, we demonstrate that one of the largest novel subunits, ATPaseTb2, is membrane-bound and localizes with monomeric and multimeric assemblies of the FoF1-ATPase. Moreover, RNAi silencing of ATPaseTb2 quickly leads to a significant decrease of the Δψm that manifests as a decreased growth phenotype, indicating that the FoF1-ATPase is impaired. To further explore the function of this protein, we employed a trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk) and thus subunit a, an essential component of the proton pore in the membrane Fo-moiety. These Dk cells generate the Δψm by combining the hydrolytic activity of the matrix-facing F1-ATPase and the electrogenic exchange of ATP4- for ADP3- by the ATP/ADP carrier (AAC). Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric FoF1-ATPase complexes were identified. In fact, the immunoprecipitation of a F1-ATPase subunit demonstrated that ATPaseTb2 was a component of these complexes. Furthermore, RNAi studies established that the membrane-bound ATPaseTb2 subunit is essential for maintaining normal growth and the Δψm of Dk cells. Thus, even in the absence of subunit a, a portion of the FoF1-ATPase is assembled in Dk cells. PMID:25714685

  1. Detection of oocyte perivitelline membrane-bound sperm: a tool for avian collection management

    PubMed Central

    Croyle, Kaitlin E.; Durrant, Barbara S.; Jensen, Thomas

    2015-01-01

    The success and sustainability of an avian breeding programme depend on managing productive and unproductive pairs. Given that each breeding season can be of immeasurable importance, it is critical to resolve pair fertility issues quickly. Such problems are traditionally diagnosed through behavioural observations, egg lay history and hatch rates, with a decision to re-pair generally taking one or more breeding seasons. In pairs producing incubated eggs that show little or no signs of embryonic development, determining fertility is difficult. Incorporating a technique to assess sperm presence on the oocyte could, in conjunction with behaviour and other data, facilitate a more timely re-pair decision. Detection of perivitelline membrane-bound (PVM-bound) sperm verifies successful copulation, sperm production and sperm functionality. Alternatively, a lack of detectable sperm, at least in freshly laid eggs, suggests no mating, lack of sperm production/function or sperm–oviduct incompatibility. This study demonstrated PVM-bound sperm detection by Hoechst staining in fresh to 24-day-incubated exotic eggs from 39 species representing 13 orders. However, a rapid and significant time-dependent loss of detectable PVM-bound sperm was observed following incubation of chicken eggs. The PCR detection of sperm in seven species, including two bacterially infected eggs, demonstrated that this method was not as reliable as visual detection using Hoechst staining. The absence of amplicons in visually positive PVMs was presumably due to large PVM size and low sperm count, resulting in DNA concentrations too low for standard PCR detection. In summary, this study demonstrated the feasibility and limitations of using PVM-bound sperm detection as a management tool for exotic avian species. We verified that sperm presence or absence on fluorescence microscopy can aid in the differentiation of fertile from infertile eggs to assist breeding managers in making prompt decisions for pair

  2. [The binuclear iron site of the membrane-bound methane hydroxylase from Methylococcus capsulatus (strain M)].

    PubMed

    Tumanova, L V; Tukhvatullin, I A; Burbaev, T Sh; Gvozdev, R I; Andersson, K K

    2008-01-01

    The particulate membrane-bound methane hydroxylase (pMMOH) was isolated from methane-oxidizing cells of Methylococcus capsulatus (strain M). At SDS PAGE, pMMOH displays three bands: 47 (alpha), 27 (beta), and 25 kDa (gamma). The ESR spectrum of pMMOH incubated with hydrogen peroxide (final concentration 20 mM) at 4 degrees C exhibited, along with the copper signal of type I with g = 2.05, signals of cytochrome with g = 3.0 and of high-spin ferriheme with g = 6.00. After incubation at -30 degrees C, additional signals with g 8.5 and 13.5 were observed. These signals, which have not been recorded previously in pMMOH preparations, are due to an intermediate of the pMMOH active site, which arises in the reaction of hydrogen peroxide with pMMOH at -30 degrees C. It was established that this intermediate is a high-spin dimer [Fe(IlI)-Fe(IV)] with S = 9/2 and different degree of rhombic distortion of structure (it is responsible for both signals). Presumably, the signal with g = 8.5 also arises from the same dimer [Fe(III)-Fe(IV)], but with S = 7/2. The presence of the intermediate [Fe(lII)-Fe(IV)] in pMMOH preparations suggests that the original state of the pMMOH active site is the dimer [Fe(III)-Fe(III)] which is located in the beta-subunit and cannot be detected by ESR. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http:// www.maik.ru. PMID:18522275

  3. Chelonian perivitelline membrane-bound sperm detection: A new breeding management tool.

    PubMed

    Croyle, Kaitlin; Gibbons, Paul; Light, Christine; Goode, Eric; Durrant, Barbara; Jensen, Thomas

    2016-03-01

    Perivitelline membrane (PVM)-bound sperm detection has recently been incorporated into avian breeding programs to assess egg fertility, confirm successful copulation, and to evaluate male reproductive status and pair compatibility. Due to the similarities between avian and chelonian egg structure and development, and because fertility determination in chelonian eggs lacking embryonic growth is equally challenging, PVM-bound sperm detection may also be a promising tool for the reproductive management of turtles and tortoises. This study is the first to successfully demonstrate the use of PVM-bound sperm detection in chelonian eggs. Recovered membranes were stained with Hoechst 33342 and examined for sperm presence using fluorescence microscopy. Sperm were positively identified for up to 206 days post-oviposition, following storage, diapause, and/or incubation, in 52 opportunistically collected eggs representing 12 species. However, advanced microbial infection frequently hindered the ability to detect membrane-bound sperm. Fertile Centrochelys sulcata, Manouria emys, and Stigmochelys pardalis eggs were used to evaluate the impact of incubation and storage on the ability to detect sperm. Storage at -20°C or in formalin were found to be the best methods for egg preservation prior to sperm detection. Additionally, sperm-derived mtDNA was isolated and PCR amplified from Astrochelys radiata, C. sulcata, and S. pardalis eggs. PVM-bound sperm detection has the potential to substantially improve studies of artificial incubation and sperm storage, and could be used to evaluate the success of artificial insemination in chelonian species. Mitochondrial DNA from PVM-bound sperm has applications for parentage analysis, the study of sperm competition, and potentially species identification. Zoo Biol. 35:95-103, 2016. © 2016 Wiley Periodicals, Inc. PMID:26890048

  4. Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration.

    PubMed

    Bardon, Clément; Poly, Franck; Piola, Florence; Pancton, Muriel; Comte, Gilles; Meiffren, Guillaume; Haichar, Feth el Zahar

    2016-05-01

    Recently, it has been shown that procyanidins from Fallopia spp. inhibit bacterial denitrification, a phenomenon called biological denitrification inhibition (BDI). However, the mechanisms involved in such a process remain unknown. Here, we investigate the mechanisms of BDI involving procyanidins, using the model strain Pseudomonas brassicacearum NFM 421. The aerobic and anaerobic (denitrification) respiration, cell permeability and cell viability of P. brassicacearum were determined as a function of procyanidin concentration. The effect of procyanidins on the bacterial membrane was observed using transmission electronic microscopy. Bacterial growth, denitrification, NO3- and NO2-reductase activity, and the expression of subunits of NO3- (encoded by the gene narG) and NO2-reductase (encoded by the gene nirS) under NO3 or NO2 were measured with and without procyanidins. Procyanidins inhibited the denitrification process without affecting aerobic respiration at low concentrations. Procyanidins also disturbed cell membranes without affecting cell viability. They specifically inhibited NO3- but not NO2-reductase.Pseudomonas brassicacearum responded to procyanidins by over-expression of the membrane-bound NO3-reductase subunit (encoded by the gene narG). Our results suggest that procyanidins can specifically inhibit membrane-bound NO3-reductase inducing enzymatic conformational changes through membrane disturbance and that P. brassicacearum responds by over-expressing membrane-bound NO3-reductase. Our results lead the way to a better understanding of BDI. PMID:26906096

  5. Mass Spectrometric Detection and Characterization of Atypical Membrane-Bound Zinc-Sensitive Phosphatases Modulating GABAA Receptors

    PubMed Central

    SidAhmed-Mezi, Mounia; Kurcewicz, Irène; Rose, Christiane; Louvel, Jacques; Sokoloff, Pierre; Pumain, René; Laschet, Jacques J.

    2014-01-01

    Background GABAA receptor (GABAAR) function is maintained by an endogenous phosphorylation mechanism for which the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the kinase. This phosphorylation is specific to the long intracellular loop I2 of the α1 subunit at two identified serine and threonine residues. The phosphorylation state is opposed by an unknown membrane-bound phosphatase, which inhibition favors the phosphorylated state of the receptor and contributes to the maintenance of its function. In cortical nervous tissue from epileptogenic areas in patients with drug-resistant epilepsies, both the endogenous phosphorylation and the functional state of the GABAAR are deficient. Methodology/Principal Findings The aim of this study is to characterize the membrane-bound phosphatases counteracting the endogenous phosphorylation of GABAAR. We have developed a new analytical tool for in vitro detection of the phosphatase activities in cortical washed membranes by liquid chromatography coupled to mass spectrometry. The substrates are two synthetic phosphopeptides, each including one of the identified endogenous phosphorylation sites of the I2 loop of GABAAR α1 subunit. We have shown the presence of multiple and atypical phosphatases sensitive to zinc ions. Patch-clamp studies of the rundown of the GABAAR currents on acutely isolated rat pyramidal cells using the phosphatase inhibitor okadaic acid revealed a clear heterogeneity of the phosphatases counteracting the function of the GABAAR. Conclusion/Significance Our results provide new insights on the regulation of GABAAR endogenous phosphorylation and function by several and atypical membrane-bound phosphatases specific to the α1 subunit of the receptor. By identifying specific inhibitors of these enzymes, novel development of antiepileptic drugs in patients with drug-resistant epilepsies may be proposed. PMID:24967814

  6. In vitro assays of three carotenogenic membrane-bound enzymes from Escherichia coli transformed with different crt genes.

    PubMed

    Fraser, P D; Sandmann, G

    1992-05-29

    In vitro assays have been developed for three membrane-bound carotenogenic enzymes, phytoene desaturase, lycopene cyclase and beta-carotene hydroxylase, expressed in Escherichia coli. Transformants of E. coli containing different deletion constructs of the Erwinia herbicola carotenogenic gene cluster were employed, allowing the estimation of enzyme activities without interference from subsequent reactions. New HPLC systems were developed to separate substrates and reaction products enabling the determination of radioactivity on-line. The newly developed assays facilitate the purification of these enzymes which have never been isolated before. PMID:1599492

  7. Modulatory Effect of Taurine on 7,12-Dimethylbenz(a)Anthracene-Induced Alterations in Detoxification Enzyme System, Membrane Bound Enzymes, Glycoprotein Profile and Proliferative Cell Nuclear Antigen in Rat Breast Tissue.

    PubMed

    Vanitha, Manickam Kalappan; Baskaran, Kuppusamy; Periyasamy, Kuppusamy; Selvaraj, Sundaramoorthy; Ilakkia, Aruldoss; Saravanan, Dhiravidamani; Venkateswari, Ramachandran; Revathi Mani, Balasundaram; Anandakumar, Pandi; Sakthisekaran, Dhanapal

    2016-08-01

    The modulatory effect of taurine on 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer in rats was studied. DMBA (25 mg/kg body weight) was administered to induce breast cancer in rats. Protein carbonyl levels, activities of membrane bound enzymes (Na(+) /K(+) ATPase, Ca(2+) ATPase, and Mg(2+) ATPase), phase I drug metabolizing enzymes (cytochrome P450, cytochrome b5, NADPH cytochrome c reductase), phase II drug metabolizing enzymes (glutathione-S-transferase and UDP-glucuronyl transferase), glycoprotein levels, and proliferative cell nuclear antigen (PCNA) were studied. DMBA-induced breast tumor bearing rats showed abnormal alterations in the levels of protein carbonyls, activities of membrane bound enzymes, drug metabolizing enzymes, glycoprotein levels, and PCNA protein expression levels. Taurine treatment (100 mg/kg body weight) appreciably counteracted all the above changes induced by DMBA. Histological examination of breast tissue further supported our biochemical findings. The results of the present study clearly demonstrated the chemotherapeutic effect of taurine in DMBA-induced breast cancer. PMID:27091720

  8. YND1, a homologue of GDA1, encodes membrane-bound apyrase required for Golgi N- and O-glycosylation in Saccharomyces cerevisiae.

    PubMed

    Gao, X D; Kaigorodov, V; Jigami, Y

    1999-07-23

    The gene for the open reading frame YER005w that is homologous to yeast Golgi GDPase encoded by the GDA1 gene was cloned and named YND1. It encodes a 630-amino acid protein that contains a single transmembrane region near the carboxyl terminus. The overexpression of the YND1 gene in the gda1 null mutant caused a significant increase in microsomal membrane-bound nucleoside phosphatase activity with a luminal orientation. The activity was equally high toward ADP/ATP, GDP/GTP, and UDP/UTP and approximately 50% less toward CDP/CTP and thiamine pyrophosphate, but there was no activity toward GMP, indicating that the Ynd1 protein belongs to the apyrase family. This substrate specificity is different from that of yeast GDPase, but similar to that of human Golgi UDPase. The Deltaynd1 mutant cells were defective in O- and N-linked glycosylation in the Golgi compartments. The overexpression of the YND1 gene complemented some glycosylation defects in Deltagda1 disruptants, suggesting a partially redundant function of yeast apyrase and GDPase. From these results and the phenotype of the Deltaynd1Deltagda1 double deletion showing a synthetic effect, we conclude that yeast apyrase is required for Golgi glycosylation and cell wall integrity, providing the first direct evidence for the in vivo function of intracellular apyrase in eukaryotic cells. PMID:10409709

  9. Mg2+ is an essential activator of hydrolytic activity of membrane-bound pyrophosphatase of Rhodospirillum rubrum.

    PubMed Central

    Sosa, A; Ordaz, H; Romero, I; Celis, H

    1992-01-01

    The substrate for the hydrolytic activity of membrane-bound pyrophosphatase is the PP(i)-Mg2+ complex. The enzyme has no activity when the free Mg2+ concentration is lower than 10 microM (at 0.5 mM-PP(i)-Mg2+), and therefore free Mg2+ is an essential activator of the hydrolytic activity. The Km for the substrate changes in response to variation in free Mg2+ concentration, from 10.25 to 0.6 mM when free Mg2+ is increased from 0.03 to 1.0 mM respectively. The Km for Mg2+ depends on the substrate concentration: the Km decreases from 0.52 to 0.14 mM from 0.25 to 0.75 mM-PP(i)-Mg2+ respectively. The extrapolated Km for Mg2+ in the absence of the substrate is 0.73 mM. Imidodiphosphate-Mg2+ and free Ca2+ were used as competitive inhibitors of substrate and activator respectively. The equilibrium binding kinetics suggest an ordered mechanism for the activator and the substrate: Mg2+ ions bind the enzyme before PP(i)-Mg2+ in the formation of the catalytic complex, membrane-bound pyrophosphatase-(Mg2+)-(PP(i)-Mg2+). PMID:1315519

  10. Widespread occurrence of N-terminal acylation in animal globins and possible origin of respiratory globins from a membrane-bound ancestor.

    PubMed

    Blank, Miriam; Burmester, Thorsten

    2012-11-01

    Proteins of the (hemo-)globin superfamily have been identified in many different animals but also occur in plants, fungi, and bacteria. Globins are renowned for their ability to store and to transport oxygen, but additional globin functions such as sensing, signaling, and detoxification have been proposed. Recently, we found that the zebrafish globin X protein is myristoylated and palmitoylated at its N-terminus. The addition of fatty acids results in an association with the cellular membranes, suggesting a previously unrecognized globin function. In this study, we show that N-terminal acylation likely occurs in globin proteins from a broad range of phyla. An N-terminal myristoylation site was identified in 90 nonredundant globins from Chlorophyta, Heterokontophyta, Cnidaria, Mollusca, Arthropoda, Nematoda, Echinodermata, Hemichordata, and Chordata (including Cephalochordata), of which 66 proteins carry an additional palmitoylation site. Bayesian phylogenetic analyses identified five major globin families, which may mirror the ancient globin diversity of the Metazoa. Globin X-like proteins form two related clades, which diverged before the radiation of the Eumetazoa. Vertebrate hemoglobin (Hb), myoglobin, cytoglobin, globin E, and globin Y form a strongly supported common clade, which is the sister group of a clade consisting of invertebrate Hbs and relatives. The N-terminally acylated globins do not form a single monophyletic group but are distributed to four distinct clades. This pattern may be either explained by multiple introduction of an N-terminal acylation site into distinct globin lineages or by the origin of animal respiratory globins from a membrane-bound ancestor. Similarly, respiratory globins were not monophyletic. This suggests that respiratory globins might have emerged independently several times and that the early metazoan globins might have been associated with a membrane and carried out a function that was related to lipid protection or

  11. Localization and Function of the Membrane-bound Riboflavin in the Na+-translocating NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae*

    PubMed Central

    Casutt, Marco S.; Huber, Tamara; Brunisholz, René; Tao, Minli; Fritz, Günter; Steuber, Julia

    2010-01-01

    The sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) from the human pathogen Vibrio cholerae is a respiratory membrane protein complex that couples the oxidation of NADH to the transport of Na+ across the bacterial membrane. The Na+-NQR comprises the six subunits NqrABCDEF, but the stoichiometry and arrangement of these subunits are unknown. Redox-active cofactors are FAD and a 2Fe-2S cluster on NqrF, covalently attached FMNs on NqrB and NqrC, and riboflavin and ubiquinone-8 with unknown localization in the complex. By analyzing the cofactor content and NADH oxidation activity of subcomplexes of the Na+-NQR lacking individual subunits, the riboflavin cofactor was unequivocally assigned to the membrane-bound NqrB subunit. Quantitative analysis of the N-terminal amino acids of the holo-complex revealed that NqrB is present in a single copy in the holo-complex. It is concluded that the hydrophobic NqrB harbors one riboflavin in addition to its covalently attached FMN. The catalytic role of two flavins in subunit NqrB during the reduction of ubiquinone to ubiquinol by the Na+-NQR is discussed. PMID:20558724

  12. Maximal Expression of Membrane-Bound Nitrate Reductase in Paracoccus Is Induced by Nitrate via a Third FNR-Like Regulator Named NarR

    PubMed Central

    Wood, Nicholas J.; Alizadeh, Tooba; Bennett, Scott; Pearce, Joanne; Ferguson, Stuart J.; Richardson, David J.; Moir, James W. B.

    2001-01-01

    Respiratory reduction of nitrate to nitrite is the first key step in the denitrification process that leads to nitrate loss from soils. In Paracoccus pantotrophus, the enzyme system that catalyzes this reaction is encoded by the narKGHJI gene cluster. Expression of this cluster is maximal under anaerobic conditions in the presence of nitrate. Upstream from narK is narR, a gene encoding a member of the FNR family of transcriptional activators. narR is transcribed divergently from the other nar genes. Mutational analysis reveals that NarR is required for maximal expression of the membrane-bound nitrate reductase genes and narK but has no other regulatory function related to denitrification. NarR is shown to require nitrate and/or nitrite is order to activate gene expression. The N-terminal region of the protein lacks the cysteine residues that are required for formation of an oxygen-sensitive iron-sulfur cluster in some other members of the FNR family. Also, NarR lacks a crucial residue involved in interactions of this family of regulators with the ς70 subunit of RNA polymerase, indicating that a different mechanism is used to promote transcription. narR is also found in Paracoccus denitrificans, indicating that this species contains at least three FNR homologues. PMID:11371524

  13. Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heat-shock transcription factor sigma 32.

    PubMed Central

    Tomoyasu, T; Gamer, J; Bukau, B; Kanemori, M; Mori, H; Rutman, A J; Oppenheim, A B; Yura, T; Yamanaka, K; Niki, H

    1995-01-01

    Escherichia coli FtsH is an essential integral membrane protein that has an AAA-type ATPase domain at its C-terminal cytoplasmic part, which is homologous to at least three ATPase subunits of the eukaryotic 26S proteasome. We report here that FtsH is involved in degradation of the heat-shock transcription factor sigma 32, a key element in the regulation of the E. coli heat-shock response. In the temperature-sensitive ftsH1 mutant, the amount of sigma 32 at a non-permissive temperature was higher than in the wild-type under certain conditions due to a reduced rate of degradation. In an in vitro system with purified components, FtsH catalyzed ATP-dependent degradation of biologically active histidine-tagged sigma 32. FtsH has a zinc-binding motif similar to the active site of zinc-metalloproteases. Protease activity of FtsH for histidine-tagged sigma 32 was stimulated by Zn2+ and strongly inhibited by the heavy metal chelating agent o-phenanthroline. We conclude that FtsH is a novel membrane-bound, ATP-dependent metalloprotease with activity for sigma 32. These findings indicate a new mechanism of gene regulation in E. coli. Images PMID:7781608

  14. Evaluation of Mut(S) and Mut⁺ Pichia pastoris strains for membrane-bound catechol-O-methyltransferase biosynthesis.

    PubMed

    Pedro, A Q; Oppolzer, D; Bonifácio, M J; Maia, C J; Queiroz, J A; Passarinha, L A

    2015-04-01

    Catechol-O-methyltransferase (COMT, EC 2.1.1.6) is an enzyme that catalyzes the methylation of catechol substrates, and while structural and functional studies of its membrane-bound isoform (MBCOMT) are still hampered by low recombinant production, Pichia pastoris has been described as an attractive host for the production of correctly folded and inserted membrane proteins. Hence, in this work, MBCOMT biosynthesis was developed using P. pastoris X33 and KM71H cells in shake flasks containing a semidefined medium with different methanol concentrations. Moreover, after P. pastoris glass beads lysis, biologically and immunologically active hMBCOMT was found mainly in the solubilized membrane fraction whose kinetic parameters were identical to its correspondent native enzyme. In addition, mixed feeds of methanol and glycerol or sorbitol were also employed, and its levels quantified using liquid chromatography coupled to refractive index detection. Overall, for the first time, two P. pastoris strains with opposite phenotypes were applied for MBCOMT biosynthesis under the control of the strongly methanol-inducible alcohol oxidase (AOX) promoter. Moreover, this eukaryotic system seems to be a promising approach to deliver MBCOMT in high quantities from fermentor cultures with a lower cost-benefit due to the cheaper cultivation media coupled with the higher titers tipically achieved in biorreactors, when compared with previously reported mammallian cell cultures. PMID:25712908

  15. In vitro assay of the chlorophyll biosynthetic enzyme Mg-chelatase: Resolution of the activity into soluble and membrane-bound fractions

    SciTech Connect

    Walker, C.J.; Weinstein, J.D. )

    1991-07-01

    The first committed step in chlorophyll synthesis is the Mg-chelatase-catalyzed insertion of magnesium into protoporphyrin IX. Since iron insertion into protoporphyrin leads to heme formation, Mg-chelatase lies at the branch point of heme and chlorophyll synthesis in chloroplasts. Little is known about the enzymology or regulation of Mg-chelatase, as it has been assayed only in intact cucumber chloroplasts. In this report we describe an in vitro assay for Mg-chelatase. Mg-chelatase activity in intact pea chloroplasts was 3- to 4-fold higher than in cucumber chloroplasts. This activity survived chloroplast lysis and could be fractionated by centrifugation into supernatant and pellet components. Both of these fractions were required to reconstitute Mg-chelatase activity, and both were inactivated by boiling indicating that the enzyme is composed of soluble and membrane-bound protein(s). The product of the reaction was confirmed fluorometrically as the magnesium chelate of the porphyrin substrate. The specific activity of the reconstituted system was typically 1 nmol of Mg-deuteroporphyrin per h per mg of protein, and activity was linear for at least 60 min under our assay conditions. ATP and magnesium were required for Mg-chelatase activity and the enzymen was sensitive to the sulfhydryl reagent N-ethylmaleimide (I{sub 50}, 20 {mu}M). Broken and reconstituted cucumber chloroplasts were unable to maintain Mg-chelatase activity. However, the cucumber supernatant fraction was active when combined with the pellet fraction of peas; the converse was not true, which suggested that the cucumber pellet was the component that lost activity during lysis.

  16. Topology of 1-Acyl-sn-glycerol-3-phosphate Acyltransferases SLC1 and ALE1 and Related Membrane-bound O-Acyltransferases (MBOATs) of Saccharomyces cerevisiae*

    PubMed Central

    Pagac, Martin; de la Mora, Hector Vazquez; Duperrex, Cécile; Roubaty, Carole; Vionnet, Christine; Conzelmann, Andreas

    2011-01-01

    In yeast, phosphatidic acid, the biosynthetic precursor for all glycerophospholipids and triacylglycerols, is made de novo by the 1-acyl-sn-glycerol-3-phosphate acyltransferases Ale1p and Slc1p. Ale1p belongs to the membrane-bound O-acyltransferase (MBOAT) family, which contains many enzymes acylating lipids but also others that acylate secretory proteins residing in the lumen of the ER. A histidine present in a very short loop between two predicted transmembrane domains is the only residue that is conserved throughout the MBOAT gene family. The yeast MBOAT proteins of known function comprise Ale1p, the ergosterol acyltransferases Are1p and Are2p, and Gup1p, the last of which acylates lysophosphatidylinositol moieties of GPI anchors on ER lumenal GPI proteins. C-terminal topology reporters added to truncated versions of Gup1p yield a topology predicting a lumenal location of its uniquely conserved histidine 447 residue. The same approach shows that Ale1p and Are2p also have the uniquely conserved histidine residing in the ER lumen. Because these data raised the possibility that phosphatidic acid could be made in the lumen of the ER, we further investigated the topology of the second yeast 1-acyl-sn-glycerol-3-phosphate acyltransferase, Slc1p. The location of C-terminal topology reporters, microsomal assays probing the protease sensitivity of inserted tags, and the accessibility of natural or artificially inserted cysteines to membrane-impermeant alkylating agents all indicate that the most conserved motif containing the presumed active site histidine of Slc1p is oriented toward the ER lumen, whereas other conserved motifs are cytosolic. The implications of these findings are discussed. PMID:21849510

  17. Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions.

    PubMed

    Azzi, Sandy; Gallerne, Cindy; Romei, Cristina; Le Coz, Vincent; Gangemi, Rosaria; Khawam, Krystel; Devocelle, Aurore; Gu, Yanhong; Bruno, Stefania; Ferrini, Silvano; Chouaib, Salem; Eid, Pierre; Azzarone, Bruno; Giron-Michel, Julien

    2015-06-01

    Intrarenal interleukin-15 (IL-15) participates to renal pathophysiology, but the role of its different membrane-bound isoforms remains to be elucidated. In this study, we reassess the biology of membrane-bound IL-15 (mb-IL-15) isoforms by comparing primary cultures of human renal proximal tubular epithelial cells (RPTEC) to peritumoral (ptumTEC), tumoral (RCC), and cancer stem cells (CSC/CD105(+)). RPTEC express a 14 to 16 kDa mb-IL-15, whose existence has been assumed but never formally demonstrated and likely represents the isoform anchored at the cell membrane through the IL-15 receptor α (IL-15Rα) chain, because it is sensitive to acidic treatment and is not competent to deliver a reverse signal. By contrast, ptumTEC, RCC, and CSC express a novel N-hyperglycosylated, short-lived transmembrane mb-IL-15 (tmb-IL-15) isoform around 27 kDa, resistant to acidic shock, delivering a reverse signal in response to its soluble receptor (sIL-15Rα). This reverse signal triggers the down-regulation of the tumor suppressor gene E-cadherin in ptumTEC and RCC but not in CSC/CD105(+), where it promotes survival. Indeed, through the AKT pathway, tmb-IL-15 protects CSC/CD105(+) from non-programmed cell death induced by serum starvation. Finally, both mb-IL-15 and tmb-IL-15 are sensitive to metalloproteases, and the cleaved tmb-IL-15 (25 kDa) displays a powerful anti-apoptotic effect on human hematopoietic cells. Overall, our data indicate that both mb-IL-15 and tmb-IL-15 isoforms play a complex role in renal pathophysiology downregulating E-cadherin and favoring cell survival. Moreover, "apparently normal" ptumTEC cells, sharing different properties with RCC, could contribute to organize an enlarged peritumoral "preneoplastic" environment committed to favor tumor progression. PMID:26152359

  18. Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus.

    PubMed

    McTernan, Patrick M; Chandrayan, Sanjeev K; Wu, Chang-Hao; Vaccaro, Brian J; Lancaster, W Andrew; Yang, Qingyuan; Fu, Dax; Hura, Greg L; Tainer, John A; Adams, Michael W W

    2014-07-11

    The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ∼ 15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na(+) ions. PMID:24860091

  19. Mitochondrial AAA proteases--towards a molecular understanding of membrane-bound proteolytic machines.

    PubMed

    Gerdes, Florian; Tatsuta, Takashi; Langer, Thomas

    2012-01-01

    Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines. PMID:22001671

  20. Membrane-bound electron transport systems of an anammox bacterium: A complexome analysis.

    PubMed

    de Almeida, Naomi M; Wessels, Hans J C T; de Graaf, Rob M; Ferousi, Christina; Jetten, Mike S M; Keltjens, Jan T; Kartal, Boran

    2016-10-01

    Electron transport, or oxidative phosphorylation, is one of the hallmarks of life. To this end, prokaryotes evolved a vast variety of protein complexes, only a small part of which have been discovered and studied. These protein complexes allow them to occupy virtually every ecological niche on Earth. Here, we applied the method of proteomics-based complexome profiling to get a better understanding of the electron transport systems of the anaerobic ammonium-oxidizing (anammox) bacteria, the N2-producing key players of the global nitrogen cycle. By this method nearly all respiratory complexes that were previously predicted from genome analysis to be involved in energy and cell carbon fixation were validated. More importantly, new and unexpected ones were discovered. We believe that complexome profiling in concert with (meta)genomics offers great opportunities to expand our knowledge on bacterial respiratory processes at a rapid and massive pace, in particular in new and thus far poorly investigated non-model and environmentally-relevant species. PMID:27461995

  1. Core Steps of Membrane-Bound Peptidoglycan Biosynthesis: Recent Advances, Insight and Opportunities

    PubMed Central

    Teo, Alvin C. K.; Roper, David I.

    2015-01-01

    We are entering an era where the efficacy of current antibiotics is declining, due to the development and widespread dispersion of antibiotic resistance mechanisms. These factors highlight the need for novel antimicrobial discovery. A large number of antimicrobial natural products elicit their effect by directly targeting discrete areas of peptidoglycan metabolism. Many such natural products bind directly to the essential cell wall precursor Lipid II and its metabolites, i.e., preventing the utlisation of vital substrates by direct binding rather than inhibiting the metabolising enzymes themselves. Concurrently, there has been an increase in the knowledge surrounding the proteins essential to the metabolism of Lipid II at and across the cytoplasmic membrane. In this review, we draw these elements together and look to future antimicrobial opportunities in this area. PMID:27025638

  2. Importance of membrane-bound catechol-O-methyltransferase in L-DOPA metabolism: a pharmacokinetic study in two types of Comt gene modified mice

    PubMed Central

    Käenmäki, M; Tammimäki, A; Garcia-Horsman, JA; Myöhänen, T; Schendzielorz, N; Karayiorgou, M; Gogos, JA; Männistö, PT

    2009-01-01

    Background and purpose: Catechol-O-methyltransferase (COMT) metabolizes compounds containing catechol structures and has two forms: soluble (S-COMT) and membrane-bound (MB-COMT). Here we report the generation of a mouse line that expresses MB-COMT but not S-COMT. We compared the effects of deleting S-COMT only or both COMT forms on the pharmacokinetics of oral L-DOPA. Experimental approach: L-DOPA (10 mg·kg−1) and carbidopa (30 mg·kg−1) were given to mice by gastric tube, and samples were taken at various times. HPLC was used to measure L-DOPA in plasma and tissue samples, and dopamine and its metabolites in brain. Immunohistochemistry and Western blotting were used to characterize the distribution of COMT protein isoforms. Key results: Lack of S-COMT did not affect the levels of L-DOPA in plasma or peripheral tissues, whereas in the full COMT-knock-out mice, these levels were increased. The levels of 3-O-methyldopa were significantly decreased in the S-COMT-deficient mice. In the brain, L-DOPA levels were not significantly increased, and dopamine was increased only in females. The total COMT activity in the S-COMT-deficient mice was 22–47% of that in the wild-type mice. In peripheral tissues, female mice had lower COMT activity than the males. Conclusions and implications: In S-COMT-deficient mice, MB-COMT in the liver and the duodenum is able to O-methylate about one-half of exogenous L-DOPA. Sexual dimorphism and activity of the two COMT isoforms seems to be tissue specific and more prominent in peripheral tissues than in the brain. PMID:19930170

  3. Identification of membrane-bound variant of metalloendopeptidase neurolysin (EC 3.4.24.16) as the non-angiotensin type 1 (non-AT1), non-AT2 angiotensin binding site.

    PubMed

    Wangler, Naomi J; Santos, Kira L; Schadock, Ines; Hagen, Fred K; Escher, Emanuel; Bader, Michael; Speth, Robert C; Karamyan, Vardan T

    2012-01-01

    Recently, we discovered a novel non-angiotensin type 1 (non-AT1), non-AT2 angiotensin binding site in rodent and human brain membranes, which is distinctly different from angiotensin receptors and key proteases processing angiotensins. It is hypothesized to be a new member of the renin-angiotensin system. This study was designed to isolate and identify this novel angiotensin binding site. An angiotensin analog, photoaffinity probe 125I-SBpa-Ang II, was used to specifically label the non-AT1, non-AT2 angiotensin binding site in mouse forebrain membranes, followed by a two-step purification procedure based on the molecular size and isoelectric point of the photoradiolabeled binding protein. Purified samples were subjected to two-dimensional gel electrophoresis followed by mass spectrometry identification of proteins in the two-dimensional gel sections containing radioactivity. LC-MS/MS analysis revealed eight protein candidates, of which the four most abundant were immunoprecipitated after photoradiolabeling. Immunoprecipitation studies indicated that the angiotensin binding site might be the membrane-bound variant of metalloendopeptidase neurolysin (EC 3.4.24.16). To verify these observations, radioligand binding and photoradiolabeling experiments were conducted in membrane preparations of HEK293 cells overexpressing mouse neurolysin or thimet oligopeptidase (EC 3.4.24.15), a closely related metalloendopeptidase of the same family. These experiments also identified neurolysin as the non-AT1, non-AT2 angiotensin binding site. Finally, brain membranes of mice lacking neurolysin were nearly devoid of the non-AT1, non-AT2 angiotensin binding site, further establishing membrane-bound neurolysin as the binding site. Future studies will focus on the functional significance of this highly specific, high affinity interaction between neurolysin and angiotensins. PMID:22039052

  4. Identification of Membrane-bound Variant of Metalloendopeptidase Neurolysin (EC 3.4.24.16) as the Non-angiotensin Type 1 (Non-AT1), Non-AT2 Angiotensin Binding Site*

    PubMed Central

    Wangler, Naomi J.; Santos, Kira L.; Schadock, Ines; Hagen, Fred K.; Escher, Emanuel; Bader, Michael; Speth, Robert C.; Karamyan, Vardan T.

    2012-01-01

    Recently, we discovered a novel non-angiotensin type 1 (non-AT1), non-AT2 angiotensin binding site in rodent and human brain membranes, which is distinctly different from angiotensin receptors and key proteases processing angiotensins. It is hypothesized to be a new member of the renin-angiotensin system. This study was designed to isolate and identify this novel angiotensin binding site. An angiotensin analog, photoaffinity probe 125I-SBpa-Ang II, was used to specifically label the non-AT1, non-AT2 angiotensin binding site in mouse forebrain membranes, followed by a two-step purification procedure based on the molecular size and isoelectric point of the photoradiolabeled binding protein. Purified samples were subjected to two-dimensional gel electrophoresis followed by mass spectrometry identification of proteins in the two-dimensional gel sections containing radioactivity. LC-MS/MS analysis revealed eight protein candidates, of which the four most abundant were immunoprecipitated after photoradiolabeling. Immunoprecipitation studies indicated that the angiotensin binding site might be the membrane-bound variant of metalloendopeptidase neurolysin (EC 3.4.24.16). To verify these observations, radioligand binding and photoradiolabeling experiments were conducted in membrane preparations of HEK293 cells overexpressing mouse neurolysin or thimet oligopeptidase (EC 3.4.24.15), a closely related metalloendopeptidase of the same family. These experiments also identified neurolysin as the non-AT1, non-AT2 angiotensin binding site. Finally, brain membranes of mice lacking neurolysin were nearly devoid of the non-AT1, non-AT2 angiotensin binding site, further establishing membrane-bound neurolysin as the binding site. Future studies will focus on the functional significance of this highly specific, high affinity interaction between neurolysin and angiotensins. PMID:22039052

  5. In vitro biosynthesis of galactans by membrane-bound galactosyltransferase from radish ( Raphanus sativus L.) seedlings.

    PubMed

    Kato, Hideaki; Takeuchi, Yoshimi; Tsumuraya, Yoichi; Hashimoto, Yohichi; Nakano, Hirofumi; Kovác, Pavol

    2003-06-01

    We investigated a galactosyltransferase (GalT) involved in the synthesis of the carbohydrate portion of arabinogalactan-proteins (AGPs), which consist of a beta-(1-->3)-galactan backbone from which consecutive (1-->6)-linked beta-Gal p residues branch off. A membrane preparation from 6-day-old primary roots of radish ( Raphanus sativus L.) transferred [(14)C]Gal from UDP-[(14)C]Gal onto a beta-(1-->3)-galactan exogenous acceptor. The reaction occurred maximally at pH 5.9-6.3 and 30 degrees C in the presence of 15 mM Mn(2+) and 0.75% Triton X-100. The apparent K(m) and V(max) values for UDP-Gal were 0.41 mM and 1,000 pmol min(-1) (mg protein)(-1), respectively. The reaction with beta-(1-->3)-galactan showed a bi-phasic kinetic character with K(m) values of 0.43 and 2.8 mg ml(-1). beta-(1-->3)-Galactooligomers were good acceptors and enzyme activity increased with increasing polymerization of Gal residues. In contrast, the enzyme was less efficient on beta-(1-->6)-oligomers. The transfer reaction for an AGP from radish mature roots was negligible but could be increased by prior enzymatic or chemical removal of alpha- l-arabinofuranose (alpha- l-Ara f) residues or both alpha- l-Ara f residues and (1-->6)-linked beta-Gal side chains. Digestion of radiolabeled products formed from beta-(1-->3)-galactan and the modified AGP with exo-beta-(1-->3)-galactanase released mainly radioactive beta-(1-->6)-galactobiose, indicating that the transfer of [(14)C]Gal occurred preferentially onto consecutive (1-->3)-linked beta-Gal chains through beta-(1-->6)-linkages, resulting in the formation of single branching points. The enzyme produced mainly a branched tetrasaccharide, Galbeta(1-->3)[Galbeta(1-->6)] Galbeta(1-->3)Gal, from beta-(1-->3)-galactotriose by incubation with UDP-Gal, confirming the preferential formation of the branching linkage. Localization of the GalT in the Golgi apparatus was revealed on a sucrose density gradient. The membrane preparation also incorporated [(14

  6. Identification of a membrane-bound transcriptional regulator that links chitin and natural competence in Vibrio cholerae.

    PubMed

    Dalia, Ankur B; Lazinski, David W; Camilli, Andrew

    2014-01-01

    Vibrio cholerae is naturally competent when grown on chitin. It is known that expression of the major regulator of competence, TfoX, is controlled by chitin; however, the molecular mechanisms underlying this requirement for chitin have remained unclear. In the present study, we identify and characterize a membrane-bound transcriptional regulator that positively regulates the small RNA (sRNA) TfoR, which posttranscriptionally enhances tfoX translation. We show that this regulation of the tfoR promoter is direct by performing electrophoretic mobility shift assays and by heterologous expression of this system in Escherichia coli. This transcriptional regulator was recently identified independently and was named "TfoS" (S. Yamamoto et al., Mol. Microbiol., in press, doi:10.1111/mmi.12462). Using a constitutively active form of TfoS, we demonstrate that the activity of this regulator is sufficient to promote competence in V. cholerae in the absence of chitin. Also, TfoS contains a large periplasmic domain, which we hypothesized interacts with chitin to regulate TfoS activity. In the heterologous host E. coli, we demonstrate that chitin oligosaccharides are sufficient to activate TfoS activity at the tfoR promoter. Collectively, these data characterize TfoS as a novel chitin-sensing transcriptional regulator that represents the direct link between chitin and natural competence in V. cholerae. IMPORTANCE Naturally competent bacteria can take up exogenous DNA from the environment and integrate it into their genome by homologous recombination. This ability to take up exogenous DNA is shared by diverse bacterial species and serves as a mechanism to acquire new genes to enhance the fitness of the organism. Several members of the family Vibrionaceae become naturally competent when grown on chitin; however, a molecular understanding of how chitin activates competence is lacking. Here, we identify a novel membrane-bound transcriptional regulator that is required for natural

  7. Structure Analysis and Conformational Transitions of the Cell Penetrating Peptide Transportan 10 in the Membrane-Bound State

    PubMed Central

    Strandberg, Erik; Verdurmen, Wouter P. R.; Bürck, Jochen; Ehni, Sebastian; Mykhailiuk, Pavel K.; Afonin, Sergii; Gerthsen, Dagmar; Komarov, Igor V.; Brock, Roland; Ulrich, Anne S.

    2014-01-01

    Structure analysis of the cell-penetrating peptide transportan 10 (TP10) revealed an exemplary range of different conformations in the membrane-bound state. The bipartite peptide (derived N-terminally from galanin and C-terminally from mastoparan) was found to exhibit prominent characteristics of (i) amphiphilic α-helices, (ii) intrinsically disordered peptides, as well as (iii) β-pleated amyloid fibrils, and these conformational states become interconverted as a function of concentration. We used a complementary approach of solid-state 19F-NMR and circular dichroism in oriented membrane samples to characterize the structural and dynamical behaviour of TP10 in its monomeric and aggregated forms. Nine different positions in the peptide were selectively substituted with either the L- or D-enantiomer of 3-(trifluoromethyl)-bicyclopent-[1.1.1]-1-ylglycine (CF3-Bpg) as a reporter group for 19F-NMR. Using the L-epimeric analogs, a comprehensive three-dimensional structure analysis was carried out in lipid bilayers at low peptide concentration, where TP10 is monomeric. While the N-terminal region is flexible and intrinsically unstructured within the plane of the lipid bilayer, the C-terminal α-helix is embedded in the membrane with an oblique tilt angle of ∼55° and in accordance with its amphiphilic profile. Incorporation of the sterically obstructive D-CF3-Bpg reporter group into the helical region leads to a local unfolding of the membrane-bound peptide. At high concentration, these helix-destabilizing C-terminal substitutions promote aggregation into immobile β-sheets, which resemble amyloid fibrils. On the other hand, the obstructive D-CF3-Bpg substitutions can be accommodated in the flexible N-terminus of TP10 where they do not promote aggregation at high concentration. The cross-talk between the two regions of TP10 thus exerts a delicate balance on its conformational switch, as the presence of the α-helix counteracts the tendency of the unfolded N

  8. Membrane-bound, pyridine nucleotide-independent L-lactate dehydrogenase of Rhodopseudomonas sphaeroides.

    PubMed Central

    Markwell, J P; Lascelles, J

    1978-01-01

    Rhodopseudomonas sphaeroides has a pyridine nucleotide-independent L-lactate dehydrogenase associated with the membrane fraction of cells grown either aerobically or phototrophically. The dehydrogenase is present in cells grown on a variety of carbon sources, but at levels less than 20% of that found in cells grown with DL-lactate. The dehydrogenase has been purified 45-fold from membranes of strain L-57, a non-photosynthetic mutant, by steps involving solubilization with lauryl dimethylamine oxide and three anion-exchange chromatography steps. The purified enzyme was specific for the L-isomer of lactate. The Km of the purified enzyme for L-lactate is 1.4 mM, whereas that of the membrane-associated enzyme is 0.5 mM. The enzyme activity was inhibited competitively by D-lactate and non-competitively by oxalate and oxamate. Quinacrine, a flavin analog, also inhibited the activity. The inducible enzyme may serve as a marker of membrane protein in studies of membrane development. PMID:304854

  9. An antibody that confers plant disease resistance targets a membrane-bound glyoxal oxidase in Fusarium.

    PubMed

    Song, Xiu-Shi; Xing, Shu; Li, He-Ping; Zhang, Jing-Bo; Qu, Bo; Jiang, Jin-He; Fan, Chao; Yang, Peng; Liu, Jin-Long; Hu, Zu-Quan; Xue, Sheng; Liao, Yu-Cai

    2016-05-01

    Plant germplasm resources with natural resistance against globally important toxigenic Fusarium are inadequate. CWP2, a Fusarium genus-specific antibody, confers durable resistance to different Fusarium pathogens that infect cereals and other crops, producing mycotoxins. However, the nature of the CWP2 target is not known. Thus, investigation of the gene coding for the CWP2 antibody target will likely provide critical insights into the mechanism underlying the resistance mediated by this disease-resistance antibody. Immunoblots and mass spectrometry analysis of two-dimensional electrophoresis gels containing cell wall proteins from Fusarium graminearum (Fg) revealed that a glyoxal oxidase (GLX) is the CWP2 antigen. Cellular localization studies showed that GLX is localized to the plasma membrane. This GLX efficiently catalyzes hydrogen peroxide production; this enzymatic activity was specifically inhibited by the CWP2 antibody. GLX-deletion strains of Fg, F. verticillioides (Fv) and F. oxysporum had significantly reduced virulence on plants. The GLX-deletion Fg and Fv strains had markedly reduced mycotoxin accumulation, and the expression of key genes in mycotoxin metabolism was downregulated. This study reveals a single gene-encoded and highly conserved cellular surface antigen that is specifically recognized by the disease-resistance antibody CWP2 and regulates both virulence and mycotoxin biosynthesis in Fusarium species. PMID:26720747

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

    PubMed Central

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

    2016-01-01

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

  11. Optical waveguide lightmode spectroscopic techniques for investigating membrane-bound ion channel activities.

    PubMed

    Székács, Inna; Kaszás, Nóra; Gróf, Pál; Erdélyi, Katalin; Szendrő, István; Mihalik, Balázs; Pataki, Agnes; Antoni, Ferenc A; Madarász, Emilia

    2013-01-01

    Optical waveguide lightmode spectroscopic (OWLS) techniques were probed for monitoring ion permeation through channels incorporated into artificial lipid environment. A novel sensor set-up was developed by depositing liposomes or cell-derived membrane fragments onto hydrophilic polytetrafluoroethylene (PTFE) membrane. The fibrous material of PTFE membrane could entrap lipoid vesicles and the water-filled pores provided environment for the hydrophilic domains of lipid-embedded proteins. The sensor surface was kept clean from the lipid holder PTFE membrane by a water- and ion-permeable polyethylene terephthalate (PET) mesh. The sensor set-up was tested with egg yolk lecithin liposomes containing gramicidin ion channels and with cell-derived membrane fragments enriched in GABA-gated anion channels. The method allowed monitoring the move of Na(+) and organic cations through gramicidin channels and detecting the Cl(-)-channel functions of the (α5β2γ2) GABAA receptor in the presence or absence of GABA and the competitive GABA-blocker bicuculline. PMID:24339925

  12. Optical Waveguide Lightmode Spectroscopic Techniques for Investigating Membrane-Bound Ion Channel Activities

    PubMed Central

    Székács, Inna; Kaszás, Nóra; Gróf, Pál; Erdélyi, Katalin; Szendrő, István; Mihalik, Balázs; Pataki, Ágnes; Antoni, Ferenc A.; Madarász, Emilia

    2013-01-01

    Optical waveguide lightmode spectroscopic (OWLS) techniques were probed for monitoring ion permeation through channels incorporated into artificial lipid environment. A novel sensor set-up was developed by depositing liposomes or cell-derived membrane fragments onto hydrophilic polytetrafluoroethylene (PTFE) membrane. The fibrous material of PTFE membrane could entrap lipoid vesicles and the water-filled pores provided environment for the hydrophilic domains of lipid-embedded proteins. The sensor surface was kept clean from the lipid holder PTFE membrane by a water- and ion-permeable polyethylene terephthalate (PET) mesh. The sensor set-up was tested with egg yolk lecithin liposomes containing gramicidin ion channels and with cell-derived membrane fragments enriched in GABA-gated anion channels. The method allowed monitoring the move of Na+ and organic cations through gramicidin channels and detecting the Cl–-channel functions of the (α5β2γ2) GABAA receptor in the presence or absence of GABA and the competitive GABA-blocker bicuculline. PMID:24339925

  13. A Novel HIV-1 Reporter Virus with a Membrane-Bound Gaussia princeps Luciferase

    PubMed Central

    Suree, Nuttee; Koizumi, Naoya; Sahakyan, Anna; Shimizu, Saki; An, Dong Sung

    2014-01-01

    Summary HIV-1 reporter viruses are a critical tool for investigating HIV-1 infection. By having a reporter gene incorporated into the HIV-1 genome, the expressed reporter protein acts as a specific tag, thus enabling specific detection of HIV-1 infected cells. Currently existing HIV-1 reporter viruses utilize reporters for the detection of HIV-1 infected cells by a single assay. A reporter virus enabling the detection of viral particles as well as HIV-1 infected cells by two assays can be more versatile for many applications. In this report, a novel reporter HIV-1 was generated by introducing a membrane-anchored form of the Gaussia princeps luciferase gene (mGluc) upstream of the nef gene in the HIV-1NL4-3 genome using a picornaviral 2A-like sequence. The resulting HIV-1NL4-3mGluc virus expresses Gaussia princeps luciferase efficiently on viral membrane and the cell surface of infected human T cell lines and primary peripheral blood mononuclear cells. This HIV-1 reporter is replication competent and the reporter gene mGluc is expressed during multiple rounds of infection. Importantly, viral particles can be detected by bioluminescence and infected cells can be detected simultaneously by bioluminescence and flow cytometric assays. With the versatility of two sensitive detection methods, this novel luciferase reporter has many applications such as cell-based screening for anti-HIV-1 agents or studies of HIV-1 pathogenicity. PMID:22483780

  14. Free and membrane-bound calcium in microgravity and microgravity effects at the membrane level

    NASA Astrophysics Data System (ADS)

    Belyavskaya, N. A.

    The changes of [Ca^2+]_i controlled is known to play a key regulatory role in numerous cellular processes especially associated with membranes. Previous studies from our laboratory have demonstrated an increase in calcium level in root cells of pea seedlings grown aboard orbital station ``Salyut 6'' /1/. These results: 1) indicate that observed Ca^2+-binding sites of membranes also consist in proteins and phospholipids; 2) suggest that such effects of space flight in membrane Ca-binding might be due to the enhancement of Ca^2+ influx through membranes. In model presented, I propose that Ca^2+-activated channels in plasma membrane in response to microgravity allow the movement of Ca^2+ into the root cells, causing a rise in cytoplasmic free Ca^2+ levels. The latter, in its turn, may induce the inhibition of a Ca^2+ efflux by Ca^2+-activated ATPases and through a Ca^2+/H^+ antiport. It is possible that increased cytosolic levels of Ca^2+ ions have stimulated hydrolysis and turnover of phosphatidylinositols, with a consequent elevation of cytosolic [Ca^2+]_i. Plant cell can response to such a Ca^2+ rise by an enhancement of membranous Ca^2+-binding activities to rescue thus a cell from an abundance of a cytotoxin. A Ca^2+-induced phase separation of membranous lipids assists to appear the structure nonstable zones with high energy level at the boundary of microdomains which are rich by some phospholipid components; there is mixing of molecules of the membranes contacted in these zones, the first stage of membranous fusion, which was found in plants exposed to microgravity. These results support the hypothesis that a target for microgravity effect is the flux mechanism of Ca^2+ to plant cell.

  15. Progesterone-induced activation of membrane-bound progesterone receptors in murine macrophage cells.

    PubMed

    Lu, Jing; Reese, Joshua; Zhou, Ying; Hirsch, Emmet

    2015-02-01

    Parturition is an inflammatory process mediated to a significant extent by macrophages. Progesterone (P4) maintains uterine quiescence in pregnancy, and a proposed functional withdrawal of P4 classically regulated by nuclear progesterone receptors (nPRs) leads to labor. P4 can affect the functions of macrophages despite the reported lack of expression of nPRs in these immune cells. Therefore, in this study we investigated the effects of the activation of the putative membrane-associated PR on the function of macrophages (a key cell for parturition) and discuss the implications of these findings for pregnancy and parturition. In murine macrophage cells (RAW 264.7), activation of mPRs by P4 modified to be active only extracellularly by conjugation to BSA (P4BSA, 1.0×10(-7) mol/l) caused a pro-inflammatory shift in the mRNA expression profile, with significant upregulation of the expression of cyclooxygenase 2 (COX2 (Ptgs2)), Il1B, and Tnf and downregulation of membrane progesterone receptor alpha (Paqr7) and oxytocin receptor (Oxtr). Pretreatment with PD98059, a MEK1/2 inhibitor, significantly reduced P4BSA-induced expression of mRNA of Il1B, Tnf, and Ptgs2. Inhibition of protein kinase A (PKA) by H89 blocked P4BSA-induced expression of Il1B and Tnf mRNA. P4BSA induced rapid phosphorylation of MEK1/2 and CREB (a downstream target of PKA). This phosphorylation was inhibited by pretreatment with PD98059 and H89, respectively, revealing that MEK1/2 and PKA are two of the components involved in mPR signaling. Taken together, these results indicate that changes in membrane progesterone receptor alpha expression and signaling in macrophages are associated with the inflammatory responses; and that these changes might contribute to the functional withdrawal of P4 related to labor. PMID:25472814

  16. Contribution of liver mitochondrial membrane-bound glutathione transferase to mitochondrial permeability transition pores

    SciTech Connect

    Hossain, Quazi Sohel; Ulziikhishig, Enkhbaatar; Lee, Kang Kwang; Yamamoto, Hideyuki; Aniya, Yoko

    2009-02-15

    We recently reported that the glutathione transferase in rat liver mitochondrial membranes (mtMGST1) is activated by S-glutathionylation and the activated mtMGST1 contributes to the mitochondrial permeability transition (MPT) pore and cytochrome c release from mitochondria [Lee, K.K., Shimoji, M., Quazi, S.H., Sunakawa, H., Aniya, Y., 2008. Novel function of glutathione transferase in rat liver mitochondrial membrane: role for cytochrome c release from mitochondria. Toxcol. Appl. Pharmacol. 232, 109-118]. In the present study we investigated the effect of reactive oxygen species (ROS), generator gallic acid (GA) and GST inhibitors on mtMGST1 and the MPT. When rat liver mitochondria were incubated with GA, mtMGST1 activity was increased to about 3 fold and the increase was inhibited with antioxidant enzymes and singlet oxygen quenchers including 1,4-diazabicyclo [2,2,2] octane (DABCO). GA-mediated mtMGST1 activation was prevented by GST inhibitors such as tannic acid, hematin, and cibacron blue and also by cyclosporin A (CsA). In addition, GA induced the mitochondrial swelling which was also inhibited by GST inhibitors, but not by MPT inhibitors CsA, ADP, and bongkrekic acid. GA also released cytochrome c from the mitochondria which was inhibited completely by DABCO, moderately by GST inhibitors, and somewhat by CsA. Ca{sup 2+}-mediated mitochondrial swelling and cytochrome c release were inhibited by MPT inhibitors but not by GST inhibitors. When the outer mitochondrial membrane was isolated after treatment of mitochondria with GA, mtMGST1 activity was markedly increased and oligomer/aggregate of mtMGST1 was observed. These results indicate that mtMGST1 in the outer mitochondrial membrane is activated by GA through thiol oxidation leading to protein oligomerization/aggregation, which may contribute to the formation of ROS-mediated, CsA-insensitive MPT pore, suggesting a novel mechanism for regulation of the MPT by mtMGST1.

  17. Crystallization and preliminary structure determination of the membrane-bound complex cytochrome c nitrite reductase from Desulfovibrio vulgaris Hildenborough

    SciTech Connect

    Rodrigues, M. L.; Oliveira, T.; Matias, P. M.; Martins, I. C.; Valente, F. M. A.; Pereira, I. A. C.; Archer, M.

    2006-06-01

    The cytochrome c nitrite reductase complex from D. vulgaris Hildenborough has been crystallized. The preliminary crystallographic structure reveals a 2:1 NrfA:NrfH complex stoichiometry. The cytochrome c nitrite reductase (cNiR) isolated from Desulfovibrio vulgaris Hildenborough is a membrane-bound complex formed of NrfA and NrfH subunits. The catalytic subunit NrfA is a soluble pentahaem cytochrome c that forms a physiological dimer of about 120 kDa. The electron-donor subunit NrfH is a membrane-anchored tetrahaem cytochrome c of about 18 kDa molecular weight and belongs to the NapC/NirT family of quinol dehydrogenases, for which no structures are known. Crystals of the native cNiR membrane complex, solubilized with dodecylmaltoside detergent (DDM), were obtained using PEG 4K as precipitant. Anomalous diffraction data were measured at the Swiss Light Source to 2.3 Å resolution. Crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 79.5, b = 256.7, c = 578.2 Å. Molecular-replacement and MAD methods were combined to solve the structure. The data presented reveal that D. vulgaris cNiR contains one NrfH subunit per NrfA dimer.

  18. Identification of a Membrane-Bound Transcriptional Regulator That Links Chitin and Natural Competence in Vibrio cholerae

    PubMed Central

    Dalia, Ankur B.; Lazinski, David W.; Camilli, Andrew

    2014-01-01

    ABSTRACT Vibrio cholerae is naturally competent when grown on chitin. It is known that expression of the major regulator of competence, TfoX, is controlled by chitin; however, the molecular mechanisms underlying this requirement for chitin have remained unclear. In the present study, we identify and characterize a membrane-bound transcriptional regulator that positively regulates the small RNA (sRNA) TfoR, which posttranscriptionally enhances tfoX translation. We show that this regulation of the tfoR promoter is direct by performing electrophoretic mobility shift assays and by heterologous expression of this system in Escherichia coli. This transcriptional regulator was recently identified independently and was named “TfoS” (S. Yamamoto et al., Mol. Microbiol., in press, doi:10.1111/mmi.12462). Using a constitutively active form of TfoS, we demonstrate that the activity of this regulator is sufficient to promote competence in V. cholerae in the absence of chitin. Also, TfoS contains a large periplasmic domain, which we hypothesized interacts with chitin to regulate TfoS activity. In the heterologous host E. coli, we demonstrate that chitin oligosaccharides are sufficient to activate TfoS activity at the tfoR promoter. Collectively, these data characterize TfoS as a novel chitin-sensing transcriptional regulator that represents the direct link between chitin and natural competence in V. cholerae. PMID:24473132

  19. A Novel Murine Anti-Lactoferrin Monoclonal Antibody Activates Human Polymorphonuclear Leukocytes through Membrane-Bound Lactoferrin and TLR4

    PubMed Central

    Hu, Xiao-Min; Xu, Yan-Rui; Yan, Ru; Sun, Shu-Liang; Dong, Hong-Liang; Wang, Jun; Gao, Xiao-Ming

    2015-01-01

    Soluble lactoferrin (LTF) is a versatile molecule that not only regulates the iron homeostasis, but also harbors direct microbicidal and immunomodulating abilities in mammalian body fluids. In contrast, little is known about the function of membrane-bound LTF (mbLTF), although its expression on human polymorphonuclear leukocytes (huPMNs) has been reported for decades. Given that LTF/anti-LTF antibodies represent a potential diagnostic/prognostic biomarker and a therapeutic target in patients with immune disorders, we wished, in the present study, to generate a novel human LTF- (huLTF-) specific mAb suitable for detailed analyses on the expression and function of mbLTF as well as for deciphering the underlying mechanisms. By using the traditional hybridoma cell fusion technology, we obtained a murine IgG1 (kappa) mAb, M-860, against huLTF. M-860 recognizes a conformational epitope of huLTF as it binds to natural, but not denatured, huLTF in ELISA. Moreover, M-860 detects mbLTF by FACS and captures endogenous huLTF in total cell lysates of huPMNs. Functionally, M-860 induces the activation of huPMNs partially through TLR4 but independently of phagocytosis. M-860 is thus a powerful tool to analyze the expression and function of human mbLTF, which will further our understanding of the roles of LTF in health and disease. PMID:26649297

  20. The effect of progesterone and 17-β estradiol on membrane-bound HLA-G in adipose derived stem cells.

    PubMed

    Moslehi, Akram; Hashemi-Beni, Batool; Moslehi, Azam; Akbari, Maryam Ali; Adib, Minoo

    2016-07-01

    Membrane-bound HLA-G (mHLA-G) discovery on adipose derived stem cells (ADSCs) as a tolerogenic and immunosuppressive molecule was very important. Many documents have shown that HLA-G expression can be controlled via some hormones such as progesterone (P4) and estradiol (E2). Therefore, this study was designed to evaluate progesterone and estradiol effects on mHLA-G in ADSCs at restricted and combination concentrations. Three independent cell lines were cultured in complete free phenol red DMEM and subcultured to achieve suffi cient cells. These cells were treated with P4, E2 and P4 plus E2 at physiologic and pregnancy concentrations for 3 days in cell culture conditions. The HLA-G positive ADSCs was measured via monoclonal anti HLA-G-FITC/MEMG-09 by means of flow cytometry in nine groups. Data were analyzed by one way ANOVA and Tukey's post hoc tests. There were no signifi cant values of the mean percentage of HLA-G positive cells in E2-treated and the combination of P4 plus E2-treated ADSCs compared to control cells (p value>0.05) but P4 had a signifi cant increase on mHLA-G in ADSCs (p value<0.05). High P4 concentration increased mHLA-G but E2 and the combination of P4 plus E2 could not change mHLA-G on ADSCs. PMID:27382350

  1. Engineering Hydrogen Gas Production from Formate in a Hyperthermophile by Heterologous Production of an 18-Subunit Membrane-bound Complex*

    PubMed Central

    Lipscomb, Gina L.; Schut, Gerrit J.; Thorgersen, Michael P.; Nixon, William J.; Kelly, Robert M.; Adams, Michael W. W.

    2014-01-01

    Biohydrogen gas has enormous potential as a source of reductant for the microbial production of biofuels, but its low solubility and poor gas mass transfer rates are limiting factors. These limitations could be circumvented by engineering biofuel production in microorganisms that are also capable of generating H2 from highly soluble chemicals such as formate, which can function as an electron donor. Herein, the model hyperthermophile, Pyrococcus furiosus, which grows optimally near 100 °C by fermenting sugars to produce H2, has been engineered to also efficiently convert formate to H2. Using a bacterial artificial chromosome vector, the 16.9-kb 18-gene cluster encoding the membrane-bound, respiratory formate hydrogen lyase complex of Thermococcus onnurineus was inserted into the P. furiosus chromosome and expressed as a functional unit. This enabled P. furiosus to utilize formate as well as sugars as an H2 source and to do so at both 80° and 95 °C, near the optimum growth temperature of the donor (T. onnurineus) and engineered host (P. furiosus), respectively. This accomplishment also demonstrates the versatility of P. furiosus for metabolic engineering applications. PMID:24318960

  2. The effect of progesterone and 17-β estradiol on membrane-bound HLA-G in adipose derived stem cells

    PubMed Central

    Moslehi, Akram; Hashemi-beni, Batool; Moslehi, Azam; Akbari, Maryam Ali

    2016-01-01

    Membrane-bound HLA-G (mHLA-G) discovery on adipose derived stem cells (ADSCs) as a tolerogenic and immunosuppressive molecule was very important. Many documents have shown that HLA-G expression can be controlled via some hormones such as progesterone (P4) and estradiol (E2). Therefore, this study was designed to evaluate progesterone and estradiol effects on mHLA-G in ADSCs at restricted and combination concentrations. Three independent cell lines were cultured in complete free phenol red DMEM and subcultured to achieve suffi cient cells. These cells were treated with P4, E2 and P4 plus E2 at physiologic and pregnancy concentrations for 3 days in cell culture conditions. The HLA-G positive ADSCs was measured via monoclonal anti HLA-G-FITC/MEMG-09 by means of flow cytometry in nine groups. Data were analyzed by one way ANOVA and Tukey's post hoc tests. There were no signifi cant values of the mean percentage of HLA-G positive cells in E2-treated and the combination of P4 plus E2-treated ADSCs compared to control cells (p value>0.05) but P4 had a signifi cant increase on mHLA-G in ADSCs (p value<0.05). High P4 concentration increased mHLA-G but E2 and the combination of P4 plus E2 could not change mHLA-G on ADSCs. PMID:27382350

  3. Neonatal transfer of membrane-bound stem cell factor improves survival and heart function in aged mice after myocardial ischemia.

    PubMed

    Sun, Zhuo; Lee, Chyan-Jang; Mejia-Guerrero, Salvador; Zhang, Yuemei; Higuchi, Koji; Li, Ren-Ke; Medin, Jeffrey A

    2012-12-01

    Stem cell mobilization to injured tissue contributes to neovascularization, resulting in regeneration after myocardial infarction (MI). We previously showed that direct cardiac injection of a recombinant lentivirus (LV) that engineers expression of membrane-bound stem cell factor (mSCF) improves outcomes immediately after MI. In this study, we evaluated the effect of neonatal LV/mSCF transduction on MI outcomes in aged mice. We constructed a recombinant LV harboring an α-myosin heavy chain promoter that drives mSCF expression and injected it into the temporal vein of neonatal mice. One year later, sustained expression of mSCF in the adult mouse hearts was detected by genomic and quantitative RT-PCR and immunohistochemistry. To evaluate the contribution of neonatal LV/mSCF delivery to recovery from MI, we induced an MI in adult LV/mSCF-transduced, LV only-transduced, and nontransduced control mice. Strikingly, LV/mSCF transduction reduced infarct scar size, enhanced angiogenesis, improved ventricular function, and significantly increased survival of the mice. Regional overexpression of CD11b, a marker of monocytes and proangiogenic cells, was observed on monocytes isolated from the infarcted hearts of LV/mSCF-transduced mice. Our data suggest a model of neonatal gene delivery that leads to sustained mSCF expression during adulthood to aid recovery from MI and prevent heart failure. PMID:22998370

  4. Stimulation of phospholipase D in rabbit platelet membranes by nucleoside triphosphates and by phosphocreatine: roles of membrane-bound GDP, nucleoside diphosphate kinase and creatine kinase.

    PubMed Central

    Fan, X T; Sherwood, J L; Haslam, R J

    1994-01-01

    Previous work has shown that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and GTP stimulate phospholipase D (PLD) in rabbit platelet membranes and that these effects are greatly enhanced by pretreatment of platelets with phorbol esters that activate protein kinase C [Van der Meulen and Haslam (1990), Biochem. J. 271, 693-700]. In the present study, the effects of Mg2+, various nucleoside triphosphates and phosphocreatine (PCr) were investigated. Platelet membranes containing phospholipids labelled with [3H]glycerol were assayed for PLD in the presence of an optimal Mg2+ concentration (10 mM) by measuring [3H]phosphatidylethanol formation in incubations that included 300 mM ethanol. In membranes from phorbolester-treated platelets, the same maximal increases in PLD activity (5-fold) were seen with 1 microM GTP[S]), and 100 microM GTP. Addition of adenosine 5'-[gamma-thio]triphosphate (ATP[S]), ITP, XTP, UTP and CTP had similar stimulatory effects, but only at > or = 1 mM. In contrast, ATP had a biphasic action, causing a maximal (2-fold) stimulation at 10 microM and smaller effects at higher concentrations; the inhibitory component of the action of ATP was blocked by 2 microM staurosporine. Guanosine 5'-[beta-thio]diphosphate decreased the stimulatory effects of ATP and ATP[S]. UDP, which can inhibit nucleoside diphosphate kinase (NDPK), decreased the activation of PLD by ATP[S], ATP, XTP, CTP and to a lesser extent ITP, but had no effect on the actions of GTP[S] and GTP. Rabbit platelet membranes contained NDPK and addition of [gamma-32P]ATP led to the formation of [32P]GTP in amounts sufficient to explain most or all of the activation of PLD; UDP prevented GTP formation. PCr (0.04-1 mM) also stimulated membrane PLD activity, an effect that was dependent on endogenous membrane-bound creatine kinase (CK). UDP and guanosine 5'-[beta-thio]diphosphate each inhibited this effect of PCr. The results show that in rabbit platelet membranes, CK, NDPK and the GTP

  5. A heteromeric membrane-bound prenyltransferase complex from hop catalyzes three sequential aromatic prenylations in the bitter acid pathway.

    PubMed

    Li, Haoxun; Ban, Zhaonan; Qin, Hao; Ma, Liya; King, Andrew J; Wang, Guodong

    2015-03-01

    Bitter acids (α and β types) account for more than 30% of the fresh weight of hop (Humulus lupulus) glandular trichomes and are well known for their contribution to the bitter taste of beer. These multiprenylated chemicals also show diverse biological activities, some of which have potential benefits to human health. The bitter acid biosynthetic pathway has been investigated extensively, and the genes for the early steps of bitter acid synthesis have been cloned and functionally characterized. However, little is known about the enzyme(s) that catalyze three sequential prenylation steps in the β-bitter acid pathway. Here, we employed a yeast (Saccharomyces cerevisiae) system for the functional identification of aromatic prenyltransferase (PT) genes. Two PT genes (HlPT1L and HlPT2) obtained from a hop trichome-specific complementary DNA library were functionally characterized using this yeast system. Coexpression of codon-optimized PT1L and PT2 in yeast, together with upstream genes, led to the production of bitter acids, but no bitter acids were detected when either of the PT genes was expressed by itself. Stepwise mutation of the aspartate-rich motifs in PT1L and PT2 further revealed the prenylation sequence of these two enzymes in β-bitter acid biosynthesis: PT1L catalyzed only the first prenylation step, and PT2 catalyzed the two subsequent prenylation steps. A metabolon formed through interactions between PT1L and PT2 was demonstrated using a yeast two-hybrid system, reciprocal coimmunoprecipitation, and in vitro biochemical assays. These results provide direct evidence of the involvement of a functional metabolon of membrane-bound prenyltransferases in bitter acid biosynthesis in hop. PMID:25564559

  6. Survival, mobility, and membrane-bound enzyme activities of freshwater planarian, Dugesia japonica, exposed to synthetic and natural surfactants.

    PubMed

    Li, Mei-Hui

    2012-04-01

    Surfactants are a major class of emerging pollutants widely used in large quantities in everyday life and commonly found in surface waters worldwide. Freshwater planarian was selected to examine the effects of different surfactants by measuring mortality, mobility, and membrane-bound enzyme activities. Among the 10 surfactants tested, the acute toxicities of betaine and polyethylene glycol (PEG-200) to planarians were relatively low, with a median lethal concentration (LC50) greater than 10,000 mg/L. The toxicity to planarians of the other eight surfactants based on 48-h LC50 could be arranged in the descending order of cetylpyridinum chloride (CPC) > 4-tert-octylphenol (4-tert-OP) > ammonium lauryl sulfate > benzalkonium chloride > saponin > sodium lauroylsarcosinate > dioctyl sulfosuccinate > dodecyl trimethyl ammonium bromide (DTAB). Both CPC and 4-tert-OP were very toxic to planarians, with 48-h LC50 values <1 mg/L. The median effective concentrations (EC50s) of planarian mobility were in the 0.1 to 50 mg/L range and were in the same range as the 24-h LC50 of planarians exposed to different surfactants, except for DTAB. In addition, significant inhibition of cholinesterase activity activities was found in planarians exposed to 4-tert-OP at 2.5 and 5 mg/L and to saponin at 10 mg/L after 2-h treatments. This result suggests that planarian mobility responses can be used as an alternative indicator for acute toxicity of surfactants after a very short exposure period. PMID:22278771

  7. Light-induced reactivation of O2-tolerant membrane-bound [Ni-Fe] hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus under turnover conditions.

    PubMed

    Ciaccafava, Alexandre; Hamon, Cyrille; Infossi, Pascale; Marchi, Valérie; Giudici-Orticoni, Marie-Thérèse; Lojou, Elisabeth

    2013-10-21

    We report the effect of UV-Vis light on the membrane-bound [Ni-Fe] hydrogenase from Aquifex aeolicus under turnover conditions. Using electrochemistry, we show a potential dependent light sensitivity and propose that a light-induced structural change of the [Ni-Fe] active site is related to an enhanced reactivation of the hydrogenase under illumination at high potentials. PMID:23999766

  8. Formation of 4-keto-D-aldopentoses and 4-pentulosonates (4-keto-D-pentonates) with unidentified membrane-bound enzymes from acetic acid bacteria.

    PubMed

    Adachi, Osao; Hours, Roque A; Shinagawa, Emiko; Akakabe, Yoshihiko; Yakushi, Toshiharu; Matsushita, Kazunobu

    2011-01-01

    In our previous study, a new microbial reaction yielding 4-keto-D-arabonate from 2,5-diketo-D-gluconate was identified with Gluconacetobacter liquefaciens RCTMR 10. It appeared that decarboxylation and dehydrogenation took place together in the reaction. To analyze the nature of the reaction, investigations were done with the membrane fraction of the organism, and 4-keto-D-arabinose was confirmed as the direct precursor of 4-keto-D-arabonate. Two novel membrane-bound enzymes, 2,5-diketo-D-gluconate decarboxylase and 4-keto-D-aldopentose 1-dehydrogenase, were involved in the reaction. Alternatively, D-arabonate was oxidized to 4-keto-D-arabonate by another membrane-bound enzyme, D-arabonate 4-dehydrogenase. More directly, D-arabinose oxidation was examined with growing cells and with the membrane fraction of G. suboxydans IFO 12528. 4-Keto-D-arabinose, the same intermediate as that from 2,5-diketo-D-gluconate, was detected, and it was oxidized to 4-keto-D-arabonate. Likewise, D-ribose was oxidized to 4-keto-D-ribose and then it was oxidized to 4-keto-D-ribonate. In addition to 4-keto-D-aldopentose 1-dehydrogenase, the presence of a novel membrane-bound enzyme, D-aldopentose 4-dehydrogenase, was confirmed in the membrane fraction. The formation of 4-keto-D-aldopentoses and 4-keto-D-pentonates (4-pentulosonates) was finally confirmed as reaction products of four different novel membrane-bound enzymes. PMID:21897028

  9. Defining a Key Receptor-CheA Kinase Contact and Elucidating Its Function in the Membrane-Bound Bacterial Chemosensory Array: A Disulfide Mapping and TAM-IDS Study

    PubMed Central

    Piasta, Kene N.; Ulliman, Caleb J.; Slivka, Peter F.; Crane, Brian R.; Falke, Joseph J.

    2013-01-01

    The three core components of the ubiquitous bacterial chemosensory array – the transmembrane chemoreceptor, the histidine kinase CheA and the adaptor protein CheW – assemble to form a membrane-bound, hexagonal lattice in which receptor transmembrane signals regulate kinase activity. Both the regulatory domain of the kinase and the adaptor protein bind to overlapping sites on the cytoplasmic tip of the receptor (termed the protein interaction region). Notably, the kinase regulatory domain (P5) and the adaptor protein share the same fold constructed of two SH3-like domains. The present study focuses on the structural interface between the receptor and the kinase regulatory domain. Two models have been proposed for this interface: Model 1 is based on the crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheW adaptor protein. This model has been used in current models of chemosensory array architecture to build the receptor-CheA kinase interface. Model 2 is based on a newly determined crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheA kinase regulatory domain. Both models present unique strengths and weaknesses, and current evidence is unable to resolve which model best describes contacts in the native chemosensory arrays of Escherichia coli, Salmonella typhimurium and other bacteria. Here we employ disulfide mapping and TAM-IDS (Tryptophan and Alanine Mutation to Identify Docking Sites) to test Models 1 and 2 in well-characterized membrane-bound arrays formed from E. coli and S. typhimurium components. The results reveal that the native array interface between the receptor protein interaction region and the kinase regulatory domain is accurately described by Model 2, but not by Model 1. In addition, the results show that the interface possesses both a structural function that contributes to stable CheA kinase binding in the array, and a regulatory function central to transmission of

  10. Polymorphisms in the Tumor Necrosis Factor Receptor Genes Affect the Expression Levels of Membrane-Bound Type I and Type II Receptors

    PubMed Central

    Sennikov, Sergey V.; Vasilyev, Filipp F.; Lopatnikova, Julia A.; Shkaruba, Nadezhda S.; Silkov, Alexander N.

    2014-01-01

    The level of TNF receptors on various cells of immune system and its association with the gene polymorphism were investigated. Determining the levels of membrane-bound TNFα receptors on peripheral blood mononuclear cells (PBMCs) was performed by flow cytometry using BD QuantiBRITE calibration particles. Soluble TNFα receptor (sTNFRs) levels were determined by ELISA and genotyping was determined by PCR-RFLP. Homozygous TT individuals at SNP −609G/T TNFRI (rs4149570) showed lower levels of sTNFRI compared to GG genotype carriers. Homozygous carriers of CC genotype at SNP −1207G/C TNFRI (rs4149569) had lower expression densities of membrane-bound TNFRI on intact CD14+ monocytes compared to individuals with the GC genotype. The frequency differences in the CD3+ and CD19+ cells expressing TNFRII in relation to SNP −1709A/T TNFRII (rs652625) in healthy individuals were also determined. The genotype CC in SNP −3609C/T TNFRII (rs590368) was associated with a lower percentage of CD14+ cells expressing TNFRII compared to individuals with the CT genotype. Patients with rheumatoid arthritis had no significant changes in the frequencies of genotypes. Reduced frequency was identified for the combination TNFRI −609GT + TNFRII −3609CC only. The polymorphisms in genes represent one of cell type-specific mechanisms affecting the expression levels of membrane-bound TNFα receptors and TNFα-mediated signaling. PMID:24782596

  11. Partial proteolysis as a probe of the conformation of the gamma subunit in activated soluble and membrane-bound chloroplast coupling factor 1.

    PubMed

    Schumann, J; Richter, M L; McCarty, R E

    1985-09-25

    Treatments that enhance the latent ATPase activity of the chloroplast coupling factor (CF1) also induce hypersensitivity of the gamma subunit toward trypsin. A number of different gamma subunit cleavage products are formed (Moroney, J. V., and McCarty, R. E. (1982) J. Biol. Chem. 257, 5910-5914). We have compared the gamma cleavage products of membrane-bound and isolated CF1, activated either by reduction of the gamma disulfide bond or by removal of the epsilon subunit. The gamma subunit of isolated CF1 lacking the epsilon subunit was cleaved to a 27,000-Da species. The same cleavage site became exposed following energy-dependent conformational changes in the membrane-bound enzyme. Activation by reduction of the gamma disulfide bond also exposed this site. However, the gamma subunit of reduced CF1 was cleaved rapidly at an additional site and trypsin treatment gave rise to a 25,000-Da gamma species. The small peptide generated by the second cleavage contains one of the cysteinyl residues of the reduced disulfide bridge of gamma. This peptide dissociates from the enzyme and can be isolated by gel filtration. The close proximity of the trypsin cleavage sites to the disulfide bond of gamma is discussed with respect to the effects of tryptic cleavage on the ATPase activity of CF1. The data indicate that structural changes in a limited region of the gamma subunit strongly influence the catalytic properties of both soluble and membrane-bound CF1. PMID:2864336

  12. Magnetic resonance and kinetic studies of the mechanism of membrane-bound sodium and potassium ion- activated adenosine triphosphatase.

    PubMed

    Grisham, C M; Mildvan, A S

    1975-01-01

    EPR and water proton relaxation rate (1/T1) studies of partially (40%) and "fully" (90%) purified preparations of membrane-bound (Na+ + K+) activated ATPase from sheep kidney indicate one tight binding site for Mn2+ per enzyme dimer, with a dissociation constant (KD = 0.88 muM) in agreement with the kinetically determined activator constant, identifying this Mn2+-binding site as the active site of the ATPase. Competition studies indicate that Mg2+ binds at this site with a dissociation constant of 1 mM in agreement with its activator constant. Inorganic phosphate and methylphosphonate bind to the enzyme-Mn2+ complex with similar high affinities and decrease 1/T1 of water protons due to a decrease from four to three in the number of rapidly exchanging water protons in the coordination sphere of enzyme-bound Mn2+. The relative effectiveness of Na+ and K+ in facilitating ternary complex formation with HPO2-4 and CH3PO2-3 as a function of pH indicates that Na+ induces the phosphate monoanion to interact with enzyme-bound Mn2+. Thus protonation of an enzyme-bound phosphoryl group would convert a K+-binding site to a Na+-binding site. Dissociation constants for K+ and Na+, estimated from NMR titrations, agreed with kinetically determined activator constants of these ions consistent with binding to the active site. Parallel 32Pi-binding studies show negligible formation (less than 7%) of a covalent E-P complex under these conditions, indicating that the NMR method has detected an additional noncovalent intermediate in ion transport. Ouabain, which increases the extent of phosphorylation of the enzyme to 24% at pH 7.8 and to 106% at pH 6.1, produced further decreases in 1/T1 of water protons. Preliminary 31P- relaxation studies of CH3PO2-3 in the presence of ATPase and Mn2+ yield an Mn to P distance (6.9 +/- 0.5 A) suggesting a second sphere enzyme-Mn-ligand-CH3PO2-3 complex. Previous kinetic studies have shown that T1+ substitutes for K+ in the activation of the enzyme

  13. Arabidopsis Type II Phosphatidylinositol 4-Kinase PI4Kγ5 Regulates Auxin Biosynthesis and Leaf Margin Development through Interacting with Membrane-Bound Transcription Factor ANAC078.

    PubMed

    Tang, Yong; Zhao, Chun-Yan; Tan, Shu-Tang; Xue, Hong-Wei

    2016-08-01

    Normal leaf margin development is important for leaf morphogenesis and contributes to diverse leaf shapes in higher plants. We here show the crucial roles of an atypical type II phosphatidylinositol 4-kinase, PI4Kγ5, in Arabidopsis leaf margin development. PI4Kγ5 presents a dynamics expression pattern along with leaf development and a T-DNA mutant lacking PI4Kγ5, pi4kγ5-1, presents serrated leaves, which is resulted from the accelerated cell division and increased auxin concentration at serration tips. Studies revealed that PI4Kγ5 interacts with and phosphorylates a membrane-bound NAC transcription factor, ANAC078. Previous studies demonstrated that membrane-bound transcription factors regulate gene transcription by undergoing proteolytic process to translocate into nucleus, and ANAC078 undergoes proteolysis by cleaving off the transmembrane region and carboxyl terminal. Western blot analysis indeed showed that ANAC078 deleting of carboxyl terminal is significantly reduced in pi4kγ5-1, indicating that PI4Kγ5 is important for the cleavage of ANAC078. This is consistent with the subcellular localization observation showing that fluorescence by GFP-ANAC078 is detected at plasma membrane but not nucleus in pi4kγ5-1 mutant and that expression of ANAC078 deleting of carboxyl terminal, driven by PI4Kγ5 promoter, could rescue the leaf serration defects of pi4kγ5-1. Further analysis showed that ANAC078 suppresses the auxin synthesis by directly binding and regulating the expression of auxin synthesis-related genes. These results indicate that PI4Kγ5 interacts with ANAC078 to negatively regulate auxin synthesis and hence influences cell proliferation and leaf development, providing informative clues for the regulation of in situ auxin synthesis and cell division, as well as the cleavage and functional mechanism of membrane-bound transcription factors. PMID:27529511

  14. Arabidopsis Type II Phosphatidylinositol 4-Kinase PI4Kγ5 Regulates Auxin Biosynthesis and Leaf Margin Development through Interacting with Membrane-Bound Transcription Factor ANAC078

    PubMed Central

    Tan, Shu-Tang; Xue, Hong-Wei

    2016-01-01

    Normal leaf margin development is important for leaf morphogenesis and contributes to diverse leaf shapes in higher plants. We here show the crucial roles of an atypical type II phosphatidylinositol 4-kinase, PI4Kγ5, in Arabidopsis leaf margin development. PI4Kγ5 presents a dynamics expression pattern along with leaf development and a T-DNA mutant lacking PI4Kγ5, pi4kγ5–1, presents serrated leaves, which is resulted from the accelerated cell division and increased auxin concentration at serration tips. Studies revealed that PI4Kγ5 interacts with and phosphorylates a membrane-bound NAC transcription factor, ANAC078. Previous studies demonstrated that membrane-bound transcription factors regulate gene transcription by undergoing proteolytic process to translocate into nucleus, and ANAC078 undergoes proteolysis by cleaving off the transmembrane region and carboxyl terminal. Western blot analysis indeed showed that ANAC078 deleting of carboxyl terminal is significantly reduced in pi4kγ5–1, indicating that PI4Kγ5 is important for the cleavage of ANAC078. This is consistent with the subcellular localization observation showing that fluorescence by GFP-ANAC078 is detected at plasma membrane but not nucleus in pi4kγ5–1 mutant and that expression of ANAC078 deleting of carboxyl terminal, driven by PI4Kγ5 promoter, could rescue the leaf serration defects of pi4kγ5–1. Further analysis showed that ANAC078 suppresses the auxin synthesis by directly binding and regulating the expression of auxin synthesis-related genes. These results indicate that PI4Kγ5 interacts with ANAC078 to negatively regulate auxin synthesis and hence influences cell proliferation and leaf development, providing informative clues for the regulation of in situ auxin synthesis and cell division, as well as the cleavage and functional mechanism of membrane-bound transcription factors. PMID:27529511

  15. A single membrane-bound enzyme catalyzes the conversion of 2,5-diketo-d-gluconate to 4-keto-d-arabonate in d-glucose oxidative fermentation by Gluconobacter oxydans NBRC 3292.

    PubMed

    Tazoe, Masaaki; Oishi, Hiromi; Kobayashi, Setsuko; Hoshino, Tatsuo

    2016-08-01

    4-Keto-d-arabonate synthase (4KAS), which converts 2,5-diketo-d-gluconate (DKGA) to 4-keto-d-arabonate (4KA) in d-glucose oxidative fermentation by some acetic acid bacteria, was solubilized from the Gluconobacter oxydans NBRC 3292 cytoplasmic membrane, and purified in an electrophoretically homogenous state. A single membrane-bound enzyme was found to catalyze the conversion from DKGA to 4KA. The 92-kDa 4KAS was a homodimeric protein not requiring O2 or a cofactor for the conversion, but was stimulated by Mn(2+). N-terminal amino acid sequencing of 4KAS, followed by gene homology search indicated a 1,197-bp open reading frame (ORF), corresponding to the GLS_c04240 locus, GenBank accession No. CP004373, encoding a 398-amino acid protein with a calculated molecular weight of 42,818 Da. An Escherichia coli transformant with the 4kas plasmid exhibited 4KAS activity. Furthermore, overexpressed recombinant 4KAS was purified in an electrophoretically homogenous state and had the same molecular size as the natural enzyme. PMID:27010909

  16. Lysophosphatidylethanolamine acyltransferase 1/membrane-bound O-acyltransferase 1 regulates morphology and function of P19C6 cell-derived neurons.

    PubMed

    Tabe, Shirou; Hikiji, Hisako; Ariyoshi, Wataru; Hashidate-Yoshida, Tomomi; Shindou, Hideo; Okinaga, Toshinori; Shimizu, Takao; Tominaga, Kazuhiro; Nishihara, Tatsuji

    2016-07-01

    Glycerophospholipids, which are components of biomembranes, are formed de novo by the Kennedy pathway and subsequently mature through the Lands cycle. Lysophospholipid acyltransferases (LPLATs) are key enzymes in both pathways and influence the fatty acid composition of biomembranes. Neuronal differentiation is characterized by neurite outgrowth, which requires biomembrane biosynthesis. However, the role of LPLATs in neuronal differentiation remains unknown. In this study, we examined whether LPLATs are involved in neuronal differentiation using all-trans-retinoic acid (ATRA)-treated P19C6 cells. In these cells, mRNA levels of lysophosphatidylethanolamine acyltransferase (LPEAT)-1/membrane-bound O-acyltransferase (MBOAT)-1 were higher than those in undifferentiated cells. LPEAT enzymatic activity increased with 16:0- and 18:1-CoA as acyl donors. When LPEAT1/MBOAT1 was knocked down with small interfering RNA (siRNA), outgrowth of neurites and expression of neuronal markers decreased in ATRA-treated P19C6 cells. Voltage-dependent calcium channel activity was also suppressed in these cells transfected with LPEAT1/MBOAT1 siRNA. These results suggest that LPEAT1/MBOAT1 plays an important role in neurite outgrowth and function.-Tabe, S., Hikiji, H., Ariyoshi, W., Hashidate-Yoshida, T., Shindou, H., Okinaga, T., Shimizu, T., Tominaga, K., Nishihara, T. Lysophosphatidylethanolamine acyltransferase 1/membrane-bound O-acyltransferase 1 regulates morphology and function of P19C6 cell-derived neurons. PMID:27048541

  17. NORE1A induction by membrane-bound CD40L (mCD40L) contributes to CD40L-induced cell death and G1 growth arrest in p21-mediated mechanism

    PubMed Central

    Elmetwali, T; Salman, A; Palmer, D H

    2016-01-01

    Membrane-bound CD40L (mCD40L) but not soluble CD40L (sCD40L) has been implicated in direct cell death induction and apoptosis in CD40-expressing carcinomas. In this study, we show that mCD40L but not sCD40L induces NORE1A/Rassf5 expression in an NFκB-dependant mechanism. NORE1A expression appeared to contribute to mCD40L-induced cell death and enhance cell transition from G1 to S phase of the cell cycle in a p21-dependent mechanism. The upregulation of p21 protein was attributed to NORE1A expression, since NORE1A inhibition resulted in p21 downregulation. p21 upregulation was concomitant with lower p53 expression in the cytoplasmic fraction with no detectable increase at the nuclear p53 level. Moreover, mCD40L-induced cell death mediated by NORE1A expression appeared to be independent of mCD40L-induced cell death mediated by sustained JNK activation since NORE1A inhibition did not affect JNK phosphorylation and vice versa. The presented data allow better understanding of the mechanism by which mCD40L induces cell death which could be exploited in the clinical development of CD40-targeted anti-cancer therapies. PMID:26986513

  18. NORE1A induction by membrane-bound CD40L (mCD40L) contributes to CD40L-induced cell death and G1 growth arrest in p21-mediated mechanism.

    PubMed

    Elmetwali, T; Salman, A; Palmer, D H

    2016-01-01

    Membrane-bound CD40L (mCD40L) but not soluble CD40L (sCD40L) has been implicated in direct cell death induction and apoptosis in CD40-expressing carcinomas. In this study, we show that mCD40L but not sCD40L induces NORE1A/Rassf5 expression in an NFκB-dependant mechanism. NORE1A expression appeared to contribute to mCD40L-induced cell death and enhance cell transition from G1 to S phase of the cell cycle in a p21-dependent mechanism. The upregulation of p21 protein was attributed to NORE1A expression, since NORE1A inhibition resulted in p21 downregulation. p21 upregulation was concomitant with lower p53 expression in the cytoplasmic fraction with no detectable increase at the nuclear p53 level. Moreover, mCD40L-induced cell death mediated by NORE1A expression appeared to be independent of mCD40L-induced cell death mediated by sustained JNK activation since NORE1A inhibition did not affect JNK phosphorylation and vice versa. The presented data allow better understanding of the mechanism by which mCD40L induces cell death which could be exploited in the clinical development of CD40-targeted anti-cancer therapies. PMID:26986513

  19. Specific release of membrane-bound annexin II and cortical cytoskeletal elements by sequestration of membrane cholesterol.

    PubMed Central

    Harder, T; Kellner, R; Parton, R G; Gruenberg, J

    1997-01-01

    Annexin II is an abundant protein which is present in the cytosol and on the cytoplasmic face of plasma membrane and early endosomes. It is generally believed that this association occurs via Ca(2+)-dependent binding to lipids, a mechanism typical for the annexin protein family. Although previous studies have shown that annexin II is involved in early endosome dynamics and organization, the precise biological role of the protein is unknown. In this study, we found that approximately 50% of the total cellular annexin was associated with membranes in a Ca(2+)-independent manner. This binding was extremely tight, since it resisted high salt and, to some extent, high pH treatments. We found, however, that membrane-associated annexin II could be quantitatively released by low concentrations of the cholesterol-sequestering agents filipin and digitonin. Both treatments released an identical and limited set of proteins but had no effects on other membrane-associated proteins. Among the released proteins, we identified, in addition to annexin II itself, the cortical cytoskeletal proteins alpha-actinin, ezrin and moesin, and membrane-associated actin. Our biochemical and immunological observations indicate that these proteins are part of a complex containing annexin II and that stability of the complex is sensitive to cholesterol sequestering agents. Since annexin II is tightly membrane-associated in a cholesterol-dependent manner, and since it seems to interact physically with elements of the cortical actin cytoskeleton, we propose that the protein serves as interface between membranes containing high amounts of cholesterol and the actin cytoskeleton. Images PMID:9188103

  20. Loss of covalently linked lipid as the mechanism for radiation-induced release of membrane-bound polysaccharide and exonuclease from Micrococcus radiodurans. [/sup 60/CO

    SciTech Connect

    Mitchel, R.E.J.

    1981-08-01

    The mechanism of ..gamma..-radiation-induced release of polysaccharide and exonuclease from the midwall membrane of Micrococcus radiodurans has been examined. These two components appear to be released independently, but by very similar processes. Direct analysis of radiation-released polysaccharide indicated the absence of an alkali-labile neutral lipid normally present in the native material. Radiation-induced release therefore probably results from the radiolytic cleavage of a covalently linked lipid which normally serves to anchor these substances to the membrane. The absence of a natural membrane-bound carotenoid had no effect on the rate of release of these components. Likewise, the absence of exonuclease in an exonuclease minus mutant did not influence the release of polysaccharide. It is suggested that the major pathway of radical transfer from the initiating .OH and culminating in the cleavage of the neutral lipid anchor may not be via the membrane.

  1. Resolution of Distinct Membrane-Bound Enzymes from Enterobacter cloacae SLD1a-1 That Are Responsible for Selective Reduction of Nitrate and Selenate Oxyanions

    PubMed Central

    Ridley, Helen; Watts, Carys A.; Richardson, David J.; Butler, Clive S.

    2006-01-01

    Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with α and β subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (αβγ) complex with an apparent molecular mass of ∼600 kDa. The individual subunit sizes are ∼100 kDa (α), ∼55 kDa (β), and ∼36 kDa (γ), with a predicted overall subunit composition of α3β3γ3. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent Km for selenate was determined to be ∼2 mM, with an observed Vmax of 500 nmol SeO42− min−1 mg−1 (kcat, ∼5.0 s−1). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase. PMID:16885262

  2. Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity.

    PubMed

    Schneider, P; Holler, N; Bodmer, J L; Hahne, M; Frei, K; Fontana, A; Tschopp, J

    1998-04-20

    Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-alpha undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-alpha) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high. PMID:9547332

  3. Effect of feeding lipids recovered from fish processing waste by lactic acid fermentation and enzymatic hydrolysis on antioxidant and membrane bound enzymes in rats.

    PubMed

    Rai, Amit Kumar; Bhaskar, N; Baskaran, V

    2015-06-01

    Fish oil recovered from fresh water fish visceral waste (FVW-FO) through lactic acid fermentation (FO-LAF) and enzymatic hydrolysis (FO-EH) were fed to rats to study their influence on lipid peroxidation and activities of antioxidant and membrane bound enzyme in liver, heart and brain. Feeding of FO-LAF and FO-EH resulted in increase (P < 0.05) in lipid peroxides level in serum, liver, brain and heart tissues compared to ground nut oil (control). Activity of catalase (40-235 %) and superoxide dismutase (17-143 %) also increased (P < 0.05) with incremental level of EPA + DHA in diet. The increase was similar to cod liver oil fed rats at same concentration of EPA + DHA. FO-LAF and FO-EH increased (P < 0.05) the Na(+)K(+) ATPase activity in liver and brain microsomes, Ca(+)Mg(+) ATPase in heart microsome and acetylcholine esterase in brain microsomes when fed with 5 % EPA + DHA. There was also significant change in fatty acid composition and cholesterol/phospholipid ratio in microsomes of rat fed with FVW-FO. Feeding FVW-FO recovered by biotechnological approaches enhanced the activity of antioxidant enzymes in tissues, modulates the activities of membrane bound enzymes and improved the fatty acid composition in microsomes of tissues similar to CLO. Utilization of these processing wastes for the production of valuable biofunctional products can reduce the mounting economic values of fish oil and minimize the environmental pollution problems. PMID:26028754

  4. The aerobic respiratory chain of the acidophilic archaeon Ferroplasma acidiphilum: A membrane-bound complex oxidizing ferrous iron.

    PubMed

    Castelle, Cindy J; Roger, Magali; Bauzan, Marielle; Brugna, Myriam; Lignon, Sabrina; Nimtz, Manfred; Golyshina, Olga V; Giudici-Orticoni, Marie-Thérèse; Guiral, Marianne

    2015-08-01

    The extremely acidophilic archaeon Ferroplasma acidiphilum is found in iron-rich biomining environments and is an important micro-organism in naturally occurring microbial communities in acid mine drainage. F. acidiphilum is an iron oxidizer that belongs to the order Thermoplasmatales (Euryarchaeota), which harbors the most extremely acidophilic micro-organisms known so far. At present, little is known about the nature or the structural and functional organization of the proteins in F. acidiphilum that impact the iron biogeochemical cycle. We combine here biochemical and biophysical techniques such as enzyme purification, activity measurements, proteomics and spectroscopy to characterize the iron oxidation pathway(s) in F. acidiphilum. We isolated two respiratory membrane protein complexes: a 850 kDa complex containing an aa3-type cytochrome oxidase and a blue copper protein, which directly oxidizes ferrous iron and reduces molecular oxygen, and a 150 kDa cytochrome ba complex likely composed of a di-heme cytochrome and a Rieske protein. We tentatively propose that both of these complexes are involved in iron oxidation respiratory chains, functioning in the so-called uphill and downhill electron flow pathways, consistent with autotrophic life. The cytochrome ba complex could possibly play a role in regenerating reducing equivalents by a reverse ('uphill') electron flow. This study constitutes the first detailed biochemical investigation of the metalloproteins that are potentially directly involved in iron-mediated energy conservation in a member of the acidophilic archaea of the genus Ferroplasma. PMID:25896560

  5. Exploiting topological constraints to reveal buried sequence motifs in the membrane-bound N-linked oligosaccharyl transferases.

    PubMed

    Jaffee, Marcie B; Imperiali, Barbara

    2011-09-01

    The central enzyme in N-linked glycosylation is the oligosaccharyl transferase (OTase), which catalyzes glycan transfer from a polyprenyldiphosphate-linked carrier to select asparagines within acceptor proteins. PglB from Campylobacter jejuni is a single-subunit OTase with homology to the Stt3 subunit of the complex multimeric yeast OTase. Sequence identity between PglB and Stt3 is low (17.9%); however, both have a similar predicted architecture and contain the conserved WWDxG motif. To investigate the relationship between PglB and other Stt3 proteins, sequence analysis was performed using 28 homologues from evolutionarily distant organisms. Since detection of small conserved motifs within large membrane-associated proteins is complicated by divergent sequences surrounding the motifs, we developed a program to parse sequences according to predicted topology and then analyze topologically related regions. This approach identified three conserved motifs that served as the basis for subsequent mutagenesis and functional studies. This work reveals that several inter-transmembrane loop regions of PglB/Stt3 contain strictly conserved motifs that are essential for PglB function. The recent publication of a 3.4 Å resolution structure of full-length C. lari OTase provides clear structural evidence that these loops play a fundamental role in catalysis [ Lizak , C. ; ( 2011 ) Nature 474 , 350 - 355 ]. The current study provides biochemical support for the role of the inter-transmembrane domain loops in OTase catalysis and demonstrates the utility of combining topology prediction and sequence analysis for exposing buried pockets of homology in large membrane proteins. The described approach allowed detection of the catalytic motifs prior to availability of structural data and reveals additional catalytically relevant residues that are not predicted by structural data alone. PMID:21812456

  6. Optimized protocol for expression and purification of membrane-bound PglB, a bacterial oligosaccharyl transferase.

    PubMed

    Jaffee, Marcie B; Imperiali, Barbara

    2013-06-01

    Asparagine-linked glycosylation (NLG) plays a significant role in a diverse range of cellular processes, including protein signaling and trafficking, the immunologic response, and immune system evasion by pathogens. A major impediment to NLG-related research is an incomplete understanding of the central enzyme in the biosynthetic pathway, the oligosaccharyl transferase (OTase). Characterization of the OTase is critical for developing ways to inhibit, engineer, and otherwise manipulate the enzyme for research and therapeutic purposes. The minimal understanding of this enzyme can be attributed to its complex, transmembrane structure, and the resulting instability and resistance to overexpression and purification. The following article describes an optimized procedure for recombinant expression and purification of PglB, a bacterial OTase, in a stably active form. The conditions screened at each step, the order of screening, and the method of comparing conditions are described. Ultimately, the following approach increased expression levels from tens of micrograms to several milligrams of active protein per liter of Escherichia coli culture, and increased stability from several hours to greater than six months post-purification. This represents the first detailed procedure for attaining a pure, active, and stable OTase in milligram quantities. In addition to presenting an optimized protocol for expression and purification of PglB, these results present a general guide for the systematic optimization of the expression, purification, and stability of a large, transmembrane protein. PMID:23583934

  7. Optimized protocol for expression and purification of membrane-bound PglB, a bacterial oligosaccharyl transferase

    PubMed Central

    Jaffee, Marcie B.; Imperiali, Barbara

    2013-01-01

    Asparagine-linked glycosylation (NLG) plays a significant role in a diverse range of cellular processes, including protein signaling and trafficking, the immunologic response, and immune system evasion by pathogens. A major impediment to NLG-related research is an incomplete understanding of the central enzyme in the biosynthetic pathway, the oligosaccharyl transferase (OTase). Characterization of the OTase is critical for developing ways to inhibit, engineer, and otherwise manipulate the enzyme for research and therapeutic purposes. The minimal understanding of this enzyme can be attributed to its complex, transmembrane structure, and the resulting instability and resistance to overexpression and purification. The following article describes an optimized procedure for recombinant expression and purification of PglB, a bacterial OTase, in a stably active form. The conditions screened at each step, the order of screening, and the method of comparing conditions are described. Ultimately, the following approach increased expression levels from tens of micrograms to several milligrams of active protein per liter of E. coli culture, and increased stability from several hours to greater than six months post-purification. This represents the first detailed procedure for attaining a pure, active, and stable OTase in milligram quantities. In addition to presenting an optimized protocol for expression and purification of PglB, these results present a general guide for the systematic optimization of the expression, purification, and stability of a large, transmembrane protein. PMID:23583934

  8. A membrane-bound matrix-metalloproteinase from Nicotiana tabacum cv. BY-2 is induced by bacterial pathogens

    PubMed Central

    Schiermeyer, Andreas; Hartenstein, Hanna; Mandal, Manoj K; Otte, Burkhard; Wahner, Verena; Schillberg, Stefan

    2009-01-01

    Background Plant matrix metalloproteinases (MMP) are conserved proteolytic enzymes found in a wide range of monocotyledonous and dicotyledonous plant species. Acting on the plant extracellular matrix, they play crucial roles in many aspects of plant physiology including growth, development and the response to stresses such as pathogen attack. Results We have identified the first tobacco MMP, designated NtMMP1, and have isolated the corresponding cDNA sequence from the tobacco suspension cell line BY-2. The overall domain structure of NtMMP1 is similar to known MMP sequences, although certain features suggest it may be constitutively active rather than dependent on proteolytic processing. The protein appears to be expressed in two forms with different molecular masses, both of which are enzymatically active as determined by casein zymography. Exchanging the catalytic domain of NtMMP1 with green fluorescent protein (GFP) facilitated subcellular localization by confocal laser scanning microscopy, showing the protein is normally inserted into the plasma membrane. The NtMMP1 gene is expressed constitutively at a low level but can be induced by exposure to bacterial pathogens. Conclusion Our biochemical analysis of NtMMP1 together with bioinformatic data on the primary sequence indicate that NtMMP1 is a constitutively-active protease. Given its induction in response to bacterial pathogens and its localization in the plasma membrane, we propose a role in pathogen defense at the cell periphery. PMID:19563670

  9. Structure of Human Annexin A6 at the Air-Water Interface and in a Membrane-Bound State

    PubMed Central

    Golczak, Marcin; Kirilenko, Aneta; Bandorowicz-Pikula, Joanna; Desbat, Bernard; Pikula, Slawomir

    2004-01-01

    We postulate the existence of a pH-sensitive domain in annexin A6 (AnxA6), on the basis of our observation of pH-dependent conformational and orientation changes of this protein and its N- (AnxA6a) and C-terminal (AnxA6b) halves in the presence of lipids. Brewster angle microscopy shows that AnxA6, AnxA6a, and AnxA6b in the absence of lipids accumulate at the air-water interface and form a stable, homogeneous layer at pH below 6.0. Under these conditions polarization modulation IR absorption spectroscopy reveals significant conformational changes of AnxA6a whereas AnxA6b preserves its α-helical structure. The orientation of protein α-helices is parallel with respect to the interface. In the presence of lipids, polarization modulation IR reflection absorption spectroscopy experiments suggest that AnxA6a incorporates into the lipid/air interface, whereas AnxA6b is adsorbed under the lipid monolayer. In this case AnxA6a regains its α-helical structures. At a higher pressure of the lipid monolayer the average orientation of the α-helices of AnxA6a changes from flat to tilted by 45° with respect to normal to the membrane interface. For AnxA6b no such changes are detected, even at a high pressure of the lipid monolayer—suggesting that the putative pH-sensitive domain of AnxA6 is localized in the N-terminal half of the protein. PMID:15298924

  10. Solubilization and Resolution of the Membrane-Bound Nitrite Reductase from Paracoccus Halodenitrificans into Nitrite and Nitric Oxide Reductases

    NASA Technical Reports Server (NTRS)

    Grant, Michael A.; Cronin, Sonja E.; Hochstein, Lawrence I.

    1984-01-01

    Membranes prepared from Paracoccus halodenitrificans reduced nitrite or nitric oxide to nitrous oxide. Extraction of these membranes with the detergent CHAPSO [3-(3-Chlolamidoporopyldimethylammonio)-1-(2- hydroxy-1-propanesulfonate)], followed by ammonium sulfate fractionation of the solubilized proteins, resulted in the separation of nitrite and nitric oxide reductase activities. The fraction containing nitrite reductase activity spectrally resembled a cd-type cytochrome. Several cytochromes were detected in the nitric oxide reductase fraction. Which, if any, of these cytochromes is associated with the reduction of nitric oxide is not clear at this time.

  11. Bismuth citrate in the quantification of inorganic phosphate and its utility in the determination of membrane-bound phosphatases.

    PubMed

    Cariani, L; Thomas, L; Brito, J; del Castillo, J R

    2004-01-01

    This paper describes a rapid and sensitive method to determine inorganic phosphate, even in the presence of labile organic phosphate compounds and large quantities of proteins. The method eliminates the use of sodium arsenite, a highly toxic compound, substituting bismuth citrate for it to stabilize the phosphomolybdic acid complex formed during the interaction of inorganic phosphate and molybdate reduced by ascorbic acid. This method has also been adapted to microplates and has been used to determine the activities of Na/K ATPase and alkaline phosphatase of intestinal basolateral and luminal plasma membranes. PMID:14654048

  12. Structural insights into the mechanism of activation of the TRPV1 channel by a membrane-bound tarantula toxin.

    PubMed

    Bae, Chanhyung; Anselmi, Claudio; Kalia, Jeet; Jara-Oseguera, Andres; Schwieters, Charles D; Krepkiy, Dmitriy; Won Lee, Chul; Kim, Eun-Hee; Kim, Jae Il; Faraldo-Gómez, José D; Swartz, Kenton J

    2016-01-01

    Venom toxins are invaluable tools for exploring the structure and mechanisms of ion channels. Here, we solve the structure of double-knot toxin (DkTx), a tarantula toxin that activates the heat-activated TRPV1 channel. We also provide improved structures of TRPV1 with and without the toxin bound, and investigate the interactions of DkTx with the channel and membranes. We find that DkTx binds to the outer edge of the external pore of TRPV1 in a counterclockwise configuration, using a limited protein-protein interface and inserting hydrophobic residues into the bilayer. We also show that DkTx partitions naturally into membranes, with the two lobes exhibiting opposing energetics for membrane partitioning and channel activation. Finally, we find that the toxin disrupts a cluster of hydrophobic residues behind the selectivity filter that are critical for channel activation. Collectively, our findings reveal a novel mode of toxin-channel recognition that has important implications for the mechanism of thermosensation. PMID:26880553

  13. Direct Observation of the Three Regions in α-Synuclein that Determine its Membrane-Bound Behaviour

    PubMed Central

    Fusco, Giuliana; De Simone, Alfonso; Tata, Gopinath; Vostrikov, Vitaly; Vendruscolo, Michele; Dobson, Christopher M.; Veglia, Gianluigi

    2014-01-01

    α-synuclein (αS) is a protein involved in neurotransmitter release in presynaptic terminals, and whose aberrant aggregation is associated with Parkinson’s disease. In dopaminergic neurons, αS exists in a tightly regulated equilibrium between water-soluble and membrane-associated forms. Here we used a combination of solid-state and solution-state NMR spectroscopy to characterize the conformations of αS bound to lipid membranes mimicking the composition and physical properties of synaptic vesicles. The study evidences three αS regions possessing distinct structural and dynamical properties, including an N-terminal helical segment having a role of membrane-anchor, an unstructured C-terminal region that is weakly associated with the membrane, and a central region acting as a sensor of the lipid properties and determining the affinity of αS membrane binding. Taken together, our data define the nature of the interactions of αS with biological membranes and provide insights into their roles in the function and in the molecular processes leading the aggregation of this protein. PMID:24871041

  14. Structural insights into the mechanism of activation of the TRPV1 channel by a membrane-bound tarantula toxin

    PubMed Central

    Bae, Chanhyung; Anselmi, Claudio; Kalia, Jeet; Jara-Oseguera, Andres; Schwieters, Charles D; Krepkiy, Dmitriy; Won Lee, Chul; Kim, Eun-Hee; Kim, Jae Il; Faraldo-Gómez, José D; Swartz, Kenton J

    2016-01-01

    Venom toxins are invaluable tools for exploring the structure and mechanisms of ion channels. Here, we solve the structure of double-knot toxin (DkTx), a tarantula toxin that activates the heat-activated TRPV1 channel. We also provide improved structures of TRPV1 with and without the toxin bound, and investigate the interactions of DkTx with the channel and membranes. We find that DkTx binds to the outer edge of the external pore of TRPV1 in a counterclockwise configuration, using a limited protein-protein interface and inserting hydrophobic residues into the bilayer. We also show that DkTx partitions naturally into membranes, with the two lobes exhibiting opposing energetics for membrane partitioning and channel activation. Finally, we find that the toxin disrupts a cluster of hydrophobic residues behind the selectivity filter that are critical for channel activation. Collectively, our findings reveal a novel mode of toxin-channel recognition that has important implications for the mechanism of thermosensation. DOI: http://dx.doi.org/10.7554/eLife.11273.001 PMID:26880553

  15. Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in Shewanella oneidensis MR-1

    SciTech Connect

    Charania, M.; Brockman, K.; Zhang, Yang; Banerjee, A.; Pinchuk, Grigoriy; Fredrickson, Jim K.; Beliaev, Alex S.; Saffarini, Daad

    2009-07-01

    Unlike other bacteria that use FNR to regulate anaerobic respiration, S. oneidensis MR-1 uses the cAMP receptor protein, CRP, for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an E. coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, DMSO, or Fe(III), whereas the deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III), and to a lesser extent with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and the cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagella biosynthesis, and electron transport, were differentially expressed in the cyaC mutant, but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration, and may contribute to additional signaling pathways independent of CRP.

  16. Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in Shewanella oneidensis MR-1

    SciTech Connect

    Charania, M.; Brockman, K. L.; Zhang, Y.; Banerjee, A.; Pinchuk, Grigoriy E.; Fredrickson, Jim K.; Beliaev, Alex S.; Saffarini, Daad

    2009-07-01

    Unlike other bacteria that use FNR to regulate anaerobic respiration, Shewanella oneidensis MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an Escherichia coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the cyaC mutant but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP.

  17. Direct observation of the three regions in α-synuclein that determine its membrane-bound behaviour

    NASA Astrophysics Data System (ADS)

    Fusco, Giuliana; de Simone, Alfonso; Gopinath, Tata; Vostrikov, Vitaly; Vendruscolo, Michele; Dobson, Christopher M.; Veglia, Gianluigi

    2014-05-01

    α-synuclein (αS) is a protein involved in neurotransmitter release in presynaptic terminals, and whose aberrant aggregation is associated with Parkinson’s disease. In dopaminergic neurons, αS exists in a tightly regulated equilibrium between water-soluble and membrane-associated forms. Here we use a combination of solid-state and solution NMR spectroscopy to characterize the conformations of αS bound to lipid membranes mimicking the composition and physical properties of synaptic vesicles. The study shows three αS regions possessing distinct structural and dynamical properties, including an N-terminal helical segment having a role of membrane anchor, an unstructured C-terminal region that is weakly associated with the membrane and a central region acting as a sensor of the lipid properties and determining the affinity of αS membrane binding. Taken together, our data define the nature of the interactions of αS with biological membranes and provide insights into their roles in the function of this protein and in the molecular processes leading to its aggregation.

  18. The weak, fluctuating, dipole moment of membrane-bound hydrogenase from Aquifex aeolicus accounts for its adaptability to charged electrodes.

    PubMed

    Oteri, Francesco; Ciaccafava, Alexandre; de Poulpiquet, Anne; Baaden, Marc; Lojou, Elisabeth; Sacquin-Mora, Sophie

    2014-06-21

    [NiFe] hydrogenases from Aquifex aeolicus (AaHase) and Desulfovibrio fructosovorans (DfHase) have been mainly studied to characterize physiological electron transfer processes, or to develop biotechnological devices such as biofuel cells. In this context, it remains difficult to control the orientation of AaHases on electrodes to achieve a fast interfacial electron transfer. Here, we study the electrostatic properties of these two proteins based on microsecond-long molecular dynamics simulations that we compare to voltammetry experiments. Our calculations show weak values and large fluctuations of the dipole direction in AaHase compared to DfHase, enabling the AaHase to absorb on both negatively and positively charged electrodes, with an orientation distribution that induces a spread in electron transfer rates. Moreover, we discuss the role of the transmembrane helix of AaHase and show that it does not substantially impact the general features of the dipole moment. PMID:24789038

  19. Ultrastructural Characterization of Turnip Mosaic Virus-Induced Cellular Rearrangements Reveals Membrane-Bound Viral Particles Accumulating in Vacuoles

    PubMed Central

    Wan, Juan; Basu, Kaustuv; Mui, Jeannie; Vali, Hojatollah; Zheng, Huanquan

    2015-01-01

    ABSTRACT Positive-strand RNA [(+) RNA] viruses remodel cellular membranes to facilitate virus replication and assembly. In the case of turnip mosaic virus (TuMV), the viral membrane protein 6K2 plays an essential role in endomembrane alterations. Although 6K2-induced membrane dynamics have been widely studied by confocal microscopy, the ultrastructure of this remodeling has not been extensively examined. In this study, we investigated the formation of TuMV-induced membrane changes by chemical fixation and high-pressure freezing/freeze substitution (HPF/FS) for transmission electron microscopy at different times of infection. We observed the formation of convoluted membranes connected to rough endoplasmic reticulum (rER) early in the infection process, followed by the production of single-membrane vesicle-like (SMVL) structures at the midstage of infection. Both SMVL and double-membrane vesicle-like structures with electron-dense cores, as well as electron-dense bodies, were found late in the infection process. Immunogold labeling results showed that the vesicle-like structures were 6K2 tagged and suggested that only the SMVL structures were viral RNA replication sites. Electron tomography (ET) was used to regenerate a three-dimensional model of these vesicle-like structures, which showed that they were, in fact, tubules. Late in infection, we observed filamentous particle bundles associated with electron-dense bodies, which suggests that these are sites for viral particle assembly. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. Our work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation. IMPORTANCE Positive-strand RNA viruses remodel cellular membranes for different stages of the infection process, such as protein translation and processing, viral RNA synthesis, particle assembly, and virus

  20. Molecular evolution of parsnip (Pastinaca sativa) membrane-bound prenyltransferases for linear and/or angular furanocoumarin biosynthesis.

    PubMed

    Munakata, Ryosuke; Olry, Alexandre; Karamat, Fazeelat; Courdavault, Vincent; Sugiyama, Akifumi; Date, Yoshiaki; Krieger, Célia; Silie, Prisca; Foureau, Emilien; Papon, Nicolas; Grosjean, Jérémy; Yazaki, Kazufumi; Bourgaud, Frédéric; Hehn, Alain

    2016-07-01

    In Apiaceae, furanocoumarins (FCs) are plant defence compounds that are present as linear or angular isomers. Angular isomers appeared during plant evolution as a protective response to herbivores that are resistant to linear molecules. Isomeric biosynthesis occurs through prenylation at the C6 or C8 position of umbelliferone. Here, we report cloning and functional characterization of two different prenyltransferases, Pastinaca sativa prenyltransferase 1 and 2 (PsPT1 and PsPT2), that are involved in these crucial reactions. Both enzymes are targeted to plastids and synthesize osthenol and demethylsuberosin (DMS) using exclusively umbelliferone and dimethylallylpyrophosphate (DMAPP) as substrates. Enzymatic characterization using heterologously expressed proteins demonstrated that PsPT1 is specialized for the synthesis of the linear form, demethylsuberosin, whereas PsPT2 more efficiently catalyses the synthesis of its angular counterpart, osthenol. These results are the first example of a complementary prenyltransferase pair from a single plant species that is involved in synthesizing defensive compounds. This study also provides a better understanding of the molecular mechanisms governing the angular FC biosynthetic pathway in apiaceous plants, which involves two paralogous enzymes that share the same phylogenetic origin. PMID:26918393

  1. Storage Protein Synthesis in Maize

    PubMed Central

    Larkins, Brian A.; Bracker, Charles E.; Tsai, C. Y.

    1976-01-01

    Undegraded free and membrane-bound polysomes were isolated from developing kernels of Zea mays L. frozen in liquid nitrogen. Freezing in liquid nitrogen was a prerequisite for preserving polysome structure in stored kernels. Membrane-bound polysomes from 22-day post-pollination kernels ground in high pH buffers containing 50 mm Mg2+ contained unique classes of large polysomes. These large polysomes were sensitive to ribonuclease, and electron micrographs verified that they were not formed by aggregation. The membrane-bound polysomes were the principal site of zein synthesis, since the major protein synthesized in vitro was similar to purified zein in its ethanol solubility and mobility on sodium dodecyl sulfate polyacrylamide gels. Images PMID:16659563

  2. The subunit structure of the follitropin (FSH) receptor. Photoaffinity labeling of the membrane-bound receptor follitropin complex in situ.

    PubMed

    Smith, R A; Branca, A A; Reichert, L E

    1985-11-15

    that the calf testis FSH receptor has a multimeric structure containing at least one 48-kDa subunit and suggest the presence of other nonidentical receptor subunit proteins. PMID:2997202

  3. Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures

    PubMed Central

    Ou, Yangguang; Wu, Juanfang; Sandberg, Mats

    2014-01-01

    This review covers recent advances in sampling fluid from the extracellular space of brain tissue by electroosmosis (EO). Two techniques, EO sampling with a single fused-silica capillary and EO push–pull perfusion, have been developed. These tools were used to investigate the function of membrane-bound enzymes with outward-facing active sites, or ectoenzymes, in modulating the activity of the neuropeptides leu-enkephalin and galanin in organotypic-hippocampal-slice cultures (OHSCs). In addition, the approach was used to determine the endogenous concentration of a thiol, cysteamine, in OHSCs. We have also investigated the degradation of coenzyme A in the extracellular space. The approach provides information on ectoenzyme activity, including Michaelis constants, in tissue, which, as far as we are aware, has not been done before. On the basis of computational evidence, EO push–pull perfusion can distinguish ectoenzyme activity with a ~100 µm spatial resolution, which is important for studies of enzyme kinetics in adjacent regions of the rat hippocampus. PMID:25168111

  4. Acute mechanical sensitization of peripheral nociceptors by aldosterone through non-genomic activation of membrane bound mineralocorticoid receptors in naive rats.

    PubMed

    Shaqura, Mohammed; Li, Xiongjuan; Al-Madol, Mohammed A; Tafelski, Sascha; Beyer-Koczorek, Antje; Mousa, Shaaban A; Schäfer, Michael

    2016-08-01

    Recently, there is increasing interest in the role of peripheral mineralocorticoid receptors (MR) to modulate pain, but their localization in neurons and glia of the periphery and their distinct involvement in pain control remains elusive. In naive Wistar rats our double immunofluorescence confocal microscopy of the spinal cord, dorsal root ganglia, sciatic nerve and innervated skin revealed that MR predominantly colocalized with calcitonin-gene-related peptide (CGRP)- and trkA-immunoreactive (IR) nociceptive neurons and only marginally with myelinated trkB-IR mechanoreceptive and trkC-IR proprioreceptive neurons underscoring a pivotal role for MR in the modulation of pain. MR could not be detected in Schwann cells, satellite cells, and astrocytes and only scarcely in spinal microglia cells excluding a relevant functional role of glia-derived MR at least in naïve rats. Intrathecal (i.t.) and intraplantar (i.pl.) application of increasing doses of the MR selective agonist aldosterone acutely increased nociceptive behavior which was reversible by a MR selective antagonist and most likely due to non-genomic effects. This was further substantiated by the first identification of membrane bound MR specific binding sites in sensory neurons of dorsal root ganglia and spinal cord. Therefore, a crucial role of MR on nociceptive neurons but not on glia cells and their impact on nociceptive behavior most likely due to immediate non-genomic effects has to be considered under normal but more so under pathological conditions in future studies. PMID:27016023

  5. Krypton Derivatization of an O2 -Tolerant Membrane-Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas Transport.

    PubMed

    Kalms, Jacqueline; Schmidt, Andrea; Frielingsdorf, Stefan; van der Linden, Peter; von Stetten, David; Lenz, Oliver; Carpentier, Philippe; Scheerer, Patrick

    2016-04-25

    [NiFe] hydrogenases are metalloenzymes catalyzing the reversible heterolytic cleavage of hydrogen into protons and electrons. Gas tunnels make the deeply buried active site accessible to substrates and inhibitors. Understanding the architecture and function of the tunnels is pivotal to modulating the feature of O2 tolerance in a subgroup of these [NiFe] hydrogenases, as they are interesting for developments in renewable energy technologies. Here we describe the crystal structure of the O2 -tolerant membrane-bound [NiFe] hydrogenase of Ralstonia eutropha (ReMBH), using krypton-pressurized crystals. The positions of the krypton atoms allow a comprehensive description of the tunnel network within the enzyme. A detailed overview of tunnel sizes, lengths, and routes is presented from tunnel calculations. A comparison of the ReMBH tunnel characteristics with crystal structures of other O2 -tolerant and O2 -sensitive [NiFe] hydrogenases revealed considerable differences in tunnel size and quantity between the two groups, which might be related to the striking feature of O2 tolerance. PMID:26913499

  6. Chronic dietary exposure to chlorpyrifos causes behavioral impairments, low activity of brain membrane-bound acetylcholinesterase, and increased brain acetylcholinesterase-R mRNA.

    PubMed

    López-Granero, Caridad; Cardona, Diana; Giménez, Estela; Lozano, Rafael; Barril, José; Sánchez-Santed, Fernando; Cañadas, Fernando

    2013-06-01

    Chlorpyrifos (CPF) is an organophosphate (OP) insecticide that is metabolically activated to the highly toxic chlorpyrifos oxon. Dietary exposure is the main route of intoxication for non-occupational exposures. However, only limited behavioral effects of chronic dietary exposure have been investigated. Therefore, male Wistar rats were fed a dose of 5mg/kg/day of CPF for thirty-one weeks. Animals were evaluated in spatial learning and impulsivity tasks after 21 weeks of CPF dietary exposure and one week after exposure ended, respectively. In addition, the degree of inhibition of brain acetylcholinesterase (AChE) was evaluated for both the soluble and particulate forms of the enzyme, as well as AChE gene expression. Also, brain acylpeptide hydrolase (APH) was investigated as an alternative target for OP-mediated effects. All variables were evaluated at various time points in response to CPF diet and after exposure ended. Results from behavioral procedures suggest cognitive and emotional disorders. Moreover, low levels of activity representing membrane-bound oligomeric forms (tetramers) were also observed. In addition, increased brain AChE-R mRNA levels were detected after four weeks of CPF dietary exposure. However, no changes in levels of brain APH were observed among groups. In conclusion, our data point to a relationship between cognitive impairments and changes in AChE forms, specifically to a high inhibition of the particulate form and a modification of alternative splicing of mRNA during CPF dietary exposure. PMID:23545134

  7. Diverse in vivo effects of soluble and membrane-bound M-CSF on tumor-associated macrophages in lymphoma xenograft model

    PubMed Central

    Liao, Jinfeng; Feng, Wenli; Wang, Rong; Ma, Shihui; Wang, Lina; Yang, Xiao; Yang, Feifei; Lin, Yongmin; Ren, Qian; Zheng, Guoguang

    2016-01-01

    Macrophage colony-stimulating factor (M-CSF) is an important cytokine for monocyte/macrophage lineage. Secretory M-CSF (sM-CSF) and membrane-bound M-CSF (mM-CSF) are two major alternative splicing isoforms. The functional diversity of these isoforms in the activation of tumor-associated macrophages (TAMs), especially in lymphoma microenvironment, has not been documented. Here, we studied the effects of M-CSF isoforms on TAMs in xenograft mouse model. More infiltrating TAMs were detected in microenvironment with mM-CSF and sM-CSF. TAMs could be divided into three subpopulations based on their expression of CD206 and Ly6C. While sM-CSF had greater potential to recruit and induce differentiation of TAMs and TAM subpopulations, mM-CSF had greater potential to induce proliferation of TAMs and TAM subpopulations. Though both isoforms educated TAMs and TAM subpopulations to M2-like macrophages, mM-CSF and sM-CSF induced different spectrums of phenotype-associated genes in TAMs and TAM subpopulations. These results suggested the diverse effects of M-CSF isoforms on the activation of TAMs and TAM subpopulations in lymphoma microenvironments. PMID:26595525

  8. Membrane-bound and soluble Fas ligands have opposite functions in photoreceptor cell death following separation from the retinal pigment epithelium

    PubMed Central

    Matsumoto, H; Murakami, Y; Kataoka, K; Notomi, S; Mantopoulos, D; Trichonas, G; Miller, J W; Gregory, M S; Ksander, B R; Marshak-Rothstein, A; Vavvas, D G

    2015-01-01

    Fas ligand (FasL) triggers apoptosis of Fas-positive cells, and previous reports described FasL-induced cell death of Fas-positive photoreceptors following a retinal detachment. However, as FasL exists in membrane-bound (mFasL) and soluble (sFasL) forms, and is expressed on resident microglia and infiltrating monocyte/macrophages, the current study examined the relative contribution of mFasL and sFasL to photoreceptor cell death after induction of experimental retinal detachment in wild-type, knockout (FasL−/−), and mFasL-only knock-in (ΔCS) mice. Retinal detachment in FasL−/− mice resulted in a significant reduction of photoreceptor cell death. In contrast, ΔCS mice displayed significantly more apoptotic photoreceptor cell death. Photoreceptor loss in ΔCS mice was inhibited by a subretinal injection of recombinant sFasL. Thus, Fas/FasL-triggered cell death accounts for a significant amount of photoreceptor cell loss following the retinal detachment. The function of FasL was dependent upon the form of FasL expressed: mFasL triggered photoreceptor cell death, whereas sFasL protected the retina, indicating that enzyme-mediated cleavage of FasL determines, in part, the extent of vision loss following the retinal detachment. Moreover, it also indicates that treatment with sFasL could significantly reduce photoreceptor cell loss in patients with retinal detachment. PMID:26583327

  9. Integrated light-scattering spectroscopy, a sensitive probe for peptide-vesicle binding: application to the membrane-bound colicin E1 channel peptide.

    PubMed Central

    Strawbridge, K. B.; Palmer, L. R.; Merrill, A. R.; Hallett, F. R.

    1995-01-01

    Integrated light-scattering (ILS) spectroscopy was used to monitor the binding of the colicin E1 channel peptide to POPC:POPG large unilamellar vesicles (LUV; 60:40, mol:mol) at acidic pH (3.5). Binding conditions were chosen such that nearly all of the channel peptide was bound to the vesicles with little free peptide remaining in solution. The increase in vesicle size upon the insertion of the channel peptide was measured by performing a discrete inversion technique on data obtained from an ILS spectrometer. Vesicle size number distributions were determined for five different systems having peptide/vesicle ratios of approximately 0, 77, 154, 206, and 257. The experiment was repeated four times (twice at two different vesicle concentrations) to determine reproducibility. The relative changes in vesicle radius upon peptide binding to the membrane vesicles was remarkably reproducible even though these changes represented only a few nanometers. A comparison of vesicle size number distributions in the absence of bound peptide was made between ILS and dynamic light scattering (DLS) data and showed similar results. However, DLS was incapable of detecting the small changes due to peptide-induced vesicle swelling. The membrane-bound volume of the colicin E1 channel peptide was approximately 177 +/- 22 nm3. These data indicate that in the absence of a membrane potential (closed channel state) the colicin E1 channel peptide inserts into the membrane resulting in a significant displacement of the lipid bilayer as evidenced from the dose-dependent increase in the vesicle radius. These results indicate that ILS spectroscopy is a sensitive sizing technique that is capable of detecting relatively small changes in membrane vesicles and may have a wide application in the determination of peptide binding to membrane vesicles. Images FIGURE 2 PMID:7711234

  10. Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases.

    PubMed

    Watanabe, Kenshi; Ohno, Makoto; Taguchi, Masahiro; Kawamoto, Seiji; Ono, Kazuhisa; Aki, Tsunehiro

    2016-01-01

    Membrane-bound desaturases are physiologically and industrially important enzymes that are involved in the production of diverse fatty acids such as polyunsaturated fatty acids and their derivatives. Here, we identified amino acid residues that determine the substrate specificity of rat Δ6 desaturase (D6d) acting on linoleoyl-CoA by comparing its amino acid sequence with that of Δ5 desaturase (D5d), which converts dihomo-γ-linolenoyl-CoA. The N-terminal cytochrome b5-like domain was excluded as a determinant by domain swapping analysis. Substitution of eight amino acid residues (Ser209, Asn211, Arg216, Ser235, Leu236, Trp244, Gln245, and Val344) of D6d with the corresponding residues of D5d by site-directed mutagenesis switched the substrate specificity from linoleoyl-CoA to dihomo-γ-linolenoyl-CoA. In addition, replacement of Leu323 of D6d with Phe323 on the basis of the amino acid sequence of zebra fish Δ5/6 bifunctional desaturase was found to render D6d bifunctional. Homology modeling of D6d using recent crystal structure data of human stearoyl-CoA (Δ9) desaturase revealed that Arg216, Trp244, Gln245, and Leu323 are located near the substrate-binding pocket. To our knowledge, this is the first report on the structural basis of the substrate specificity of a mammalian front-end fatty acid desaturase, which will aid in efficient production of value-added fatty acids. PMID:26590171

  11. A functionally critical single nucleotide polymorphism in the gene encoding the membrane-bound alcohol dehydrogenase found in ethanol oxidation-deficient Gluconobacter thailandicus.

    PubMed

    Charoenyingcharoen, Piyanat; Matsutani, Minenosuke; Yakushi, Toshiharu; Theeragool, Gunjana; Yukphan, Pattaraporn; Matsushita, Kazunobu

    2015-08-10

    The Gluconobacter thailandicus strains NBRC3254, NBRC3255, NBRC3256, NBRC3257, and NBRC3258 are naturally deficient in the ethanol-oxidizing respiratory chain because they do not produce the cytochrome subunit of the membrane-bound alcohol dehydrogenase (ADH). Draft genomes of G. thailandicus strains NBRC3255 and NBRC3257 indicated that the adhB gene encoding the cytochrome subunit contains four base differences when compared to a closely related gene in the public database One of the nucleotide differences results in an Opal codon at the -19th tryptophan (Trp) in the signal sequence for translocation to the periplasmic space (here, the position of +1st residue is assigned to the N-terminal amino acid residue after signal peptide cleavage), while the other differences result in one missense and two silent amino acid alterations. All five of the G. thailandicus strains were shown to have the Trp(-19)Opal alteration. Ethanol oxidation and ADH activities in NBRC3255 were restored by transformation with a derivative of the endogenous adhB gene, of which the -19th Opal codon was altered to encode Trp. These results indicate that this sequence is a functionally critical single nucleotide polymorphism in the cytochrome subunit. Comparative genomic analyses between the draft genomes of NBRC3255 and NBRC3257 revealed that although the two genomes are closely related, they both have a significant number of unique open reading frames. We suggest that the closely related NBRC3255 and NBRC3257 diverged from a common ancestor having the mutation in the adhB gene, whereas no additional functionally critical mutation occurred in the adhB pseudogene over the course of evolution. PMID:25943635

  12. A Heteromeric Membrane-Bound Prenyltransferase Complex from Hop Catalyzes Three Sequential Aromatic Prenylations in the Bitter Acid Pathway1[OPEN

    PubMed Central

    Li, Haoxun; Ban, Zhaonan; Qin, Hao; Ma, Liya; King, Andrew J.

    2015-01-01

    Bitter acids (α and β types) account for more than 30% of the fresh weight of hop (Humulus lupulus) glandular trichomes and are well known for their contribution to the bitter taste of beer. These multiprenylated chemicals also show diverse biological activities, some of which have potential benefits to human health. The bitter acid biosynthetic pathway has been investigated extensively, and the genes for the early steps of bitter acid synthesis have been cloned and functionally characterized. However, little is known about the enzyme(s) that catalyze three sequential prenylation steps in the β-bitter acid pathway. Here, we employed a yeast (Saccharomyces cerevisiae) system for the functional identification of aromatic prenyltransferase (PT) genes. Two PT genes (HlPT1L and HlPT2) obtained from a hop trichome-specific complementary DNA library were functionally characterized using this yeast system. Coexpression of codon-optimized PT1L and PT2 in yeast, together with upstream genes, led to the production of bitter acids, but no bitter acids were detected when either of the PT genes was expressed by itself. Stepwise mutation of the aspartate-rich motifs in PT1L and PT2 further revealed the prenylation sequence of these two enzymes in β-bitter acid biosynthesis: PT1L catalyzed only the first prenylation step, and PT2 catalyzed the two subsequent prenylation steps. A metabolon formed through interactions between PT1L and PT2 was demonstrated using a yeast two-hybrid system, reciprocal coimmunoprecipitation, and in vitro biochemical assays. These results provide direct evidence of the involvement of a functional metabolon of membrane-bound prenyltransferases in bitter acid biosynthesis in hop. PMID:25564559

  13. Membrane Bending by Protein Crowding

    NASA Astrophysics Data System (ADS)

    Stachowiak, Jeanne

    2014-03-01

    From endosomes and synaptic vesicles to the cristae of the mitochondria and the annulus of the nuclear pore, highly curved membranes are fundamental to the structure and physiology of living cells. The established view is that specific families of proteins are able to bend membranes by binding to them. For example, inherently curved proteins are thought to impose their structure on the membrane surface, while membrane-binding proteins with hydrophobic motifs are thought to insert into the membrane like wedges, driving curvature. However, computational models have recently revealed that these mechanisms would require specialized membrane-bending proteins to occupy nearly 100% of a curved membrane surface, an improbable physiological situation given the immense density and diversity of membrane-bound proteins, and the low expression levels of these specialized proteins within curved regions of the membrane. How then does curvature arise within the complex and crowded environment of cellular membranes? Our recent work using proteins involved in clathrin-mediated endocytosis, as well as engineered protein-lipid interactions, has suggested a new hypothesis - that lateral pressure generated by collisions between membrane-bound proteins can drive membrane bending. Specifically, by correlating membrane bending with quantitative optical measurements of protein density on synthetic membrane surfaces and simple physical models of collisions among membrane-bound proteins, we have demonstrated that protein-protein steric interactions can drive membrane curvature. These findings suggest that a simple imbalance in the concentration of membrane-bound proteins across a membrane surface can drive a membrane to bend, providing an efficient mechanism by which essentially any protein can contribute to shaping membranes.

  14. Growth of the Obligate Anaerobe Desulfovibrio vulgaris Hildenborough under Continuous Low Oxygen Concentration Sparging: Impact of the Membrane-Bound Oxygen Reductases

    PubMed Central

    Ramel, Fanny; Brasseur, Gael; Pieulle, Laetitia; Valette, Odile; Hirschler-Réa, Agnès; Fardeau, Marie Laure; Dolla, Alain

    2015-01-01

    Although obligate anaerobe, the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) exhibits high aerotolerance that involves several enzymatic systems, including two membrane-bound oxygen reductases, a bd-quinol oxidase and a cc(b/o)o3 cytochrome oxidase. Effect of constant low oxygen concentration on growth and morphology of the wild-type, single (Δbd, Δcox) and double deletion (Δcoxbd) mutant strains of the genes encoding these oxygen reductases was studied. When both wild-type and deletion mutant strains were cultured in lactate/sulfate medium under constant 0.02% O2 sparging, they were able to grow but the final biomasses and the growth yield were lower than that obtained under anaerobic conditions. At the end of the growth, lactate was not completely consumed and when conditions were then switched to anaerobic, growth resumed. Time-lapse microscopy revealed that a large majority of the cells were then able to divide (over 97%) but the time to recover a complete division event was longer for single deletion mutant Δbd than for the three other strains. Determination of the molar growth yields on lactate suggested that a part of the energy gained from lactate oxidation was derived toward cells protection/repairing against oxidative conditions rather than biosynthesis, and that this part was higher in the single deletion mutant Δbd and, to a lesser extent, Δcox strains. Our data show that when DvH encounters oxidative conditions, it is able to stop growing and to rapidly resume growing when conditions are switched to anaerobic, suggesting that it enters active dormancy sate under oxidative conditions. We propose that the pyruvate-ferredoxin oxidoreductase (PFOR) plays a central role in this phenomenon by reversibly switching from an oxidative-sensitive fully active state to an oxidative-insensitive inactive state. The oxygen reductases, and especially the bd-quinol oxidase, would have a crucial function by maintaining reducing conditions

  15. Membrane-Bound CYB5R3 Is a Common Effector of Nutritional and Oxidative Stress Response Through FOXO3a and Nrf2

    PubMed Central

    Siendones, Emilio; SantaCruz-Calvo, Sara; Martín-Montalvo, Alejandro; Cascajo, María V.; Ariza, Julia; López-Lluch, Guillermo; Villalba, José M.; Acquaviva-Bourdain, Cécile; Roze, Emmanuel; Bernier, Michel; de Cabo, Rafael

    2014-01-01

    Abstract Aims: Membrane-bound CYB5R3 deficiency in humans causes recessive hereditary methaemoglobinaemia (RHM), an incurable disease that is characterized by severe neurological disorders. CYB5R3 encodes for NADH-dependent redox enzyme that contributes to metabolic homeostasis and stress protection; however, how it is involved in the neurological pathology of RHM remains unknown. Here, the role and transcriptional regulation of CYB5R3 was studied under nutritional and oxidative stress. Results: CYB5R3-deficient cells exhibited a decrease of the NAD+/NADH ratio, mitochondrial respiration rate, ATP production, and mitochondrial electron transport chain activities, which were associated with higher sensitivity to oxidative stress, and an increase in senescence-associated β-galactosidase activity. Overexpression of either forkhead box class O 3a (FOXO3a) or nuclear factor (erythroid-derived 2)-like2 (Nrf2) was associated with increased CYB5R3 levels, and genetic ablation of Nrf2 resulted in lower CYB5R3 expression. The presence of two antioxidant response element sequences in the CYB5R3 promoter led to chromatin immunoprecipitation studies, which showed that cellular stressors enhanced the binding of Nrf2 and FOXO3a to the CYB5R3 promoter. Innovation: Our findings demonstrate that CYB5R3 contributes to regulate redox homeostasis, aerobic metabolism, and cellular senescence, suggesting that CYB5R3 might be a key effector of oxidative and nutritional stress pathways. The expression of CYB5R3 is regulated by the cooperation of Nrf2 and FOXO3a. Conclusion: CYB5R3 is an essential gene that appears as a final effector for both nutritional and oxidative stress responses through FOXO3a and Nrf2, respectively, and their interaction promotes CYB5R3 expression. These results unveil a potential mechanism of action by which CYB5R3 deficiency contributes to the pathophysiological underpinnings of neurological disorders in RHM patients. Antioxid. Redox Signal. 21, 1708–1725. PMID

  16. TUNABLE TENSOR VOTING FOR REGULARIZING PUNCTATE PATTERNS OFMEMBRANE-BOUND PROTEIN SIGNALS

    SciTech Connect

    Loss, Leandro; Bebis, George; Parvin, Bahram

    2009-04-29

    Membrane-bound protein, expressed in the basal-lateral region, is heterogeneous and an important endpoint for understanding biological processes. At the optical resolution, membrane-bound protein can be visualized as being diffused (e.g., E-cadherin), punctate (e.g., connexin), or simultaneously diffused and punctate as a result of sample preparation or conditioning. Furthermore, there is a significant amount of heterogeneity as a result of technical and biological variations. This paper aims at enhancing membrane-bound proteins that are expressed between epithelial cells so that quantitative analysis can be enabled on a cell-by-cell basis. We propose a method to detect and enhance membrane-bound protein signal from noisy images. More precisely, we build upon the tensor voting framework in order to produce an efficient method to detect and refine perceptually interesting linear structures in images. The novelty of the proposed method is in its iterative tuning of the tensor voting fields, which allows the concentration of the votes only over areas of interest. The method is shown to produce high quality enhancements of membrane-bound protein signals with combined punctate and diffused characteristics. Experimental results demonstrate the benefits of using tunable tensor voting for enhancing and differentiating cell-cell adhesion mediated by integral cell membrane protein.

  17. Two unrelated putative membrane-bound progestin receptors, progesterone membrane receptor component 1 (PGMRC1) and membrane progestin receptor (mPR) beta, are expressed in the rainbow trout oocyte and exhibit similar ovarian expression patterns

    PubMed Central

    Mourot, Brigitte; Nguyen, Thaovi; Fostier, Alexis; Bobe, Julien

    2006-01-01

    Background In lower vertebrates, steroid-induced oocyte maturation is considered to involve membrane-bound progestin receptors. Two totally distinct classes of putative membrane-bound progestin receptors have been reported in vertebrates. A first class of receptors, now termed progesterone membrane receptor component (PGMRC; subtypes 1 and 2) has been studied since 1996 but never studied in a fish species nor in the oocyte of any animal species. A second class of receptors, termed membrane progestin receptors (mPR; subtypes alpha, beta and gamma), was recently described in vertebrates and implicated in the progestin-initiated induction of oocyte maturation in fish. Methods In the present study, we report the characterization of the full coding sequence of rainbow trout PGMRC1 and mPR beta cDNAs, their tissue distribution, their ovarian expression profiles during oogenesis, their hormonal regulation in the full grown ovary and the in situ localization of PGMRC1 mRNA in the ovary. Results Our results clearly show, for the first time in any animal species, that rainbow trout PGMRC1 mRNA is present in the oocyte and has a strong expression in ovarian tissue. In addition, we show that both mPR beta and PGMRC1, two members of distinct membrane-bound progestin receptor classes, exhibit highly similar ovarian expression profiles during the reproductive cycle with maximum levels during vitellogenesis and a down-expression during late vitellogenesis. In addition, the mRNA abundance of both genes is not increased after in vitro hormonal stimulation of full grown follicles by maturation inducing hormones. Conclusion Together, our findings suggest that PGMRC1 is a new possible participant in the progestin-induced oocyte maturation in fish. However, its participation in the process of oocyte maturation, which remains to be confirmed, would occur at post-transcriptional levels. PMID:16457725

  18. Towards Co-Evolution of Membrane Proteins and Metabolism

    NASA Astrophysics Data System (ADS)

    Wilson, Michael A.; Wei, Chenyu; Pohorille, Andrew

    2014-12-01

    Primordial metabolism co-evolved with the earliest membrane peptides to produce more environmentally fit progeny. Here, we map a continuous, evolutionary path that connects nascent biochemistry with simple, membrane-bound oligopeptides, ion channels and, further, membrane proteins capable of energy transduction and utilization of energy for active transport.

  19. OXIDATIVE STRESS ACTIVATES ANION EXCHANGE PROTEIN 2 AND AP-1 IN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Anion exchange protein 2 (AE2) is a membrane-bound protein that mediates chloride-bicarbonate exchange. In addition to regulating intracellular pH and cell volume, AE2 exports superoxide (O.) to the extracellular matrix in an HCO-dependent process. Given this ability to export O....

  20. Aqueous Fraction of Beta vulgaris Ameliorates Hyperglycemia in Diabetic Mice due to Enhanced Glucose Stimulated Insulin Secretion, Mediated by Acetylcholine and GLP-1, and Elevated Glucose Uptake via Increased Membrane Bound GLUT4 Transporters

    PubMed Central

    Kabir, Ashraf Ul; Samad, Mehdi Bin; Ahmed, Arif; Jahan, Mohammad Rajib; Akhter, Farjana; Tasnim, Jinat; Hasan, S. M. Nageeb; Sayfe, Sania Sarker; Hannan, J. M. A.

    2015-01-01

    Background The study was designed to investigate the probable mechanisms of anti-hyperglycemic activity of B. Vulgaris. Methodology/Principal Findings Aqueous fraction of B. Vulgaris extract was the only active fraction (50mg/kg). Plasma insulin level was found to be the highest at 30 mins after B. Vulgaris administration at a dose of 200mg/kg. B. Vulgaris treated mice were also assayed for plasma Acetylcholine, Glucagon Like Peptide-1 (GLP-1), Gastric Inhibitory Peptide (GIP), Vasoactive Intestinal Peptide, Pituitary Adenylate Cyclase-Activating Peptide (PACAP), Insulin Like Growth Factor-1 (IGF-1), Pancreatic Polypeptides (PP), and Somatostatin, along with the corresponding insulin levels. Plasma Acetylcholine and GLP-1 significantly increased in B. Vulgaris treated animals and were further studied. Pharmacological enhancers, inhibitors, and antagonists of Acetylcholine and GLP-1 were also administered to the test animals, and corresponding insulin levels were measured. These studies confirmed the role of acetylcholine and GLP-1 in enhanced insulin secretion (p<0.05). Principal signaling molecules were quantified in isolated mice islets for the respective pathways to elucidate their activities. Elevated concentrations of Acetylcholine and GLP-1 in B. Vulgaris treated mice were found to be sufficient to activate the respective pathways for insulin secretion (p<0.05). The amount of membrane bound GLUT1 and GLUT4 transporters were quantified and the subsequent glucose uptake and glycogen synthesis were assayed. We showed that levels of membrane bound GLUT4 transporters, glucose-6-phosphate in skeletal myocytes, activity of glycogen synthase, and level of glycogen deposited in the skeletal muscles all increased (p<0.05). Conclusion Findings of the present study clearly prove the role of Acetylcholine and GLP-1 in the Insulin secreting activity of B. Vulgaris. Increased glucose uptake in the skeletal muscles and subsequent glycogen synthesis may also play a part in

  1. Copper deficiency decreases the protein expression of Complex IV but not Complex I, II, III, or V in mitochondrial respiratory chain in rat heart

    Technology Transfer Automated Retrieval System (TEKTRAN)

    It has been documented that dietary copper (Cu) deficiency impairs mitochondrial respiratory function which is catalyzed by five membrane-bound multiple protein complexes. However, there are few reports on the simultaneous analysis of Cu effect on the subunit protein expression on all five protein c...

  2. Differential effects of vasopressin and phenylephrine on protein kinase C-mediated protein phosphorylations in isolated hepatocytes

    SciTech Connect

    Cooper, R.H.; Johanson, R.A.; Wiliamson, J.R.

    1986-05-01

    Receptor-mediated breakdown of inositol lipids produces two intracellular signals, diacylglycerol, which activates protein kinase C, and inositol trisphosphate, which causes release of intracellular vesicular Ca/sup 2 +/. This study examined the effects of Ca/sup 2 +/-ionophores, vasopressin, phenylephrine, and phorbol ester (PMA) on hepatocyte protein phosphorylations. (/sup 32/P) Phosphoproteins from hepatocytes prelabeled with /sup 32/P were resolved by 2-dimensional SDS-PAGE and corresponding autoradiographs were quantitated by densitometric analysis. The phosphorylation of five proteins, a plasma membrane bound 16 kDa protein with pI 6.4, a cytosolic 16 kDa protein with pI 5.8, and proteins with Mr's of 36 kDa, 52 kDa, and 68 kDa, could be attributed to phosphorylation by protein kinase C since the phosphorylation was stimulated by PMA. When the vasopressin concentration was varied, low vasopressin stimulated the phosphorylation of only the membrane bound 16 kDa protein of the above set of proteins, while higher vasopressin concentrations were required to stimulate the phosphorylation of all five proteins. Phenylephrine, even at supramaximal concentrations, stimulated the phosphorylation of only the membrane bound 16 kDa protein. These results suggest that phenylephrine is a less potent activator of protein kinase C than vasopressin by virtue of limited or localized diacylglycerol production.

  3. Evaluation of a purification procedure for the muscarinic receptor for the purpose of quantitative receptor assays of anticholinergics. Part A: The membrane-bound receptor.

    PubMed

    Smisterová, J; Ensing, K; de Zeeuw, R A

    1995-11-01

    The presented purification procedure for the muscarinic receptor from calf striatum includes the extraction of lipids with hexane in the first step and the removal of 39% of non-receptor proteins with 2 M NaCl in the second step. The simplicity of such an approach to the purification of the receptor warrants its use in the routine practice for quantitative purposes. The high affinity binding of tertiary 3H-dexetimide (3H-DEX) and quaternary 3H-N-methylscopolamine (3H-NMS) is preserved after the removal of irrelevant lipids and proteins from the P2-pellet. The overall yield of receptors--80%, when labelled with 3H-NMS, was satisfactory. Moreover, the final product, the NaCl-pellet, exerts a higher density of 3H-NMS binding sites per mg proteins by a factor of about 1.7. The overall yield of receptors and purification factor were lower, when measured with 3H-DEX. The total yield of 3H-DEX binding sites amounted to about 40% and the receptor density per mg protein decreased by a factor of 0.85. We did not succeed in the improvement of the ratio specific/non-specific binding, neither for 3H-DEX nor for 3H-NMS for the purified receptor preparations. The use of 3H-NMS is preferable to 3H-DEX in plasma sample assays because of a negligible effect of plasma on ligand binding when compared with 3H-DEX. PMID:8570569

  4. The MS Risk Allele of CD40 Is Associated with Reduced Cell-Membrane Bound Expression in Antigen Presenting Cells: Implications for Gene Function

    PubMed Central

    Field, Judith; Shahijanian, Fernando; Schibeci, Stephen; Johnson, Laura; Gresle, Melissa; Laverick, Louise; Parnell, Grant; Stewart, Graeme; McKay, Fiona; Kilpatrick, Trevor; Butzkueven, Helmut; Booth, David

    2015-01-01

    Human genetic and animal studies have implicated the costimulatory molecule CD40 in the development of multiple sclerosis (MS). We investigated the cell specific gene and protein expression variation controlled by the CD40 genetic variant(s) associated with MS, i.e. the T-allele at rs1883832. Previously we had shown that the risk allele is expressed at a lower level in whole blood, especially in people with MS. Here, we have defined the immune cell subsets responsible for genotype and disease effects on CD40 expression at the mRNA and protein level. In cell subsets in which CD40 is most highly expressed, B lymphocytes and dendritic cells, the MS-associated risk variant is associated with reduced CD40 cell-surface protein expression. In monocytes and dendritic cells, the risk allele additionally reduces the ratio of expression of full-length versus truncated CD40 mRNA, the latter encoding secreted CD40. We additionally show that MS patients, regardless of genotype, express significantly lower levels of CD40 cell-surface protein compared to unaffected controls in B lymphocytes. Thus, both genotype-dependent and independent down-regulation of cell-surface CD40 is a feature of MS. Lower expression of a co-stimulator of T cell activation, CD40, is therefore associated with increased MS risk despite the same CD40 variant being associated with reduced risk of other inflammatory autoimmune diseases. Our results highlight the complexity and likely individuality of autoimmune pathogenesis, and could be consistent with antiviral and/or immunoregulatory functions of CD40 playing an important role in protection from MS. PMID:26068105

  5. Formation of the N-N bond from nitric oxide by a membrane-bound cytochrome bc complex of nitrate-respiring (denitrifying) Pseudomonas stutzeri

    SciTech Connect

    Heiss, B.; Frunzke, K.; Zumft, W.G. )

    1989-06-01

    Nitric oxide (NO) reductase was solubilized by Triton X-100 from the membrane fraction of Pseudomonas stutzeri ZoBell and purified 100-fold to apparent electrophoretic homogeneity. The enzyme consisted of two polypeptides of M{sub r} 38,000 and 17,000 associated with heme b and heme c, respectively. Absorption maxima of the reduced complex were at 420.5, 522.5, and 552.5 nm, with a shoulder at 560 nm. The electron paramagnetic resonance spectrum was characteristic of high- and low-spin ferric heme proteins; no signals typical for iron-sulfur proteins were found. Nitric oxide reductase stoichiometrically transformed NO to nitrous oxide in an ascorbate-phenazine methosulfate-dependent reaction with a specific activity of 11.8 {mu}mol/min per mg of protein. The activity increased to 40 {mu}mol upon the addition of soybean phospholipids, n-octyl-{beta}-D-glucopyranoside, or its thio derivative to the assay system. Apparent K{sub m} values for NO and phenazine methosulfate were 60 and 2 {mu}M, respectively. The pH optimum of the reaction was at 4.8 Cytochrome co was purified from P. stutzeri to permit its distinction from NO reductase. Spectrophotometric binding assays and other criteria also differentiated NO reductase from the respiratory cytochrome bc{sub 1} complex.

  6. Membrane-bound MinDE complex acts as a toggle switch that drives Min oscillation coupled to cytoplasmic depletion of MinD

    PubMed Central

    Vecchiarelli, Anthony G.; Li, Min; Mizuuchi, Michiyo; Hwang, Ling Chin; Seol, Yeonee; Neuman, Keir C.; Mizuuchi, Kiyoshi

    2016-01-01

    The Escherichia coli Min system self-organizes into a cell-pole to cell-pole oscillator on the membrane to prevent divisions at the cell poles. Reconstituting the Min system on a lipid bilayer has contributed to elucidating the oscillatory mechanism. However, previous in vitro patterns were attained with protein densities on the bilayer far in excess of those in vivo and failed to recapitulate the standing wave oscillations observed in vivo. Here we studied Min protein patterning at limiting MinD concentrations reflecting the in vivo conditions. We identified “burst” patterns—radially expanding and imploding binding zones of MinD, accompanied by a peripheral ring of MinE. Bursts share several features with the in vivo dynamics of the Min system including standing wave oscillations. Our data support a patterning mechanism whereby the MinD-to-MinE ratio on the membrane acts as a toggle switch: recruiting and stabilizing MinD on the membrane when the ratio is high and releasing MinD from the membrane when the ratio is low. Coupling this toggle switch behavior with MinD depletion from the cytoplasm drives a self-organized standing wave oscillator. PMID:26884160

  7. A Membrane-bound eIF2 Alpha Kinase Located in Endosomes Is Regulated by Heme and Controls Differentiation and ROS Levels in Trypanosoma cruzi

    PubMed Central

    da Silva Augusto, Leonardo; Moretti, Nilmar Silvio; Ramos, Thiago Cesar Prata; de Jesus, Teresa Cristina Leandro; Zhang, Min; Castilho, Beatriz A.; Schenkman, Sergio

    2015-01-01

    Translation initiation has been described as a key step for the control of growth and differentiation of several protozoan parasites in response to environmental changes. This occurs by the activation of protein kinases that phosphorylate the alpha subunit of the translation initiation factor 2 (eIF2α), which decreases translation, and in higher eukaryotes favors the expression of stress remedial response genes. However, very little is known about the signals that activate eIF2α kinases in protozoan parasites. Here, we characterized an eIF2α kinase of Trypanosoma cruzi (TcK2), the agent of Chagas’ disease, as a transmembrane protein located in organelles that accumulate nutrients in proliferating parasite forms. We found that heme binds specifically to the catalytic domain of the kinase, inhibiting its activity. In the absence of heme, TcK2 is activated, arresting cell growth and inducing differentiation of proliferative into infective and non-proliferative forms. Parasites lacking TcK2 lose this differentiation capacity and heme is not stored in reserve organelles, remaining in the cytosol. TcK2 null cells display growth deficiencies, accumulating hydrogen peroxide that drives the generation of reactive oxygen species. The augmented level of hydrogen peroxide occurs as a consequence of increased superoxide dismutase activity and decreased peroxide activity. These phenotypes could be reverted by the re-expression of the wild type but not of a TcK2 dead mutant. These findings indicate that heme is a key factor for the growth control and differentiation through regulation of an unusual type of eIF2α kinase in T. cruzi. PMID:25658109

  8. Association of cytochrome c with membrane-bound cytochrome c oxidase proceeds parallel to the membrane rather than in bulk solution.

    PubMed

    Spaar, Alexander; Flöck, Dagmar; Helms, Volkhard

    2009-03-01

    Electron transfer between the water-soluble cytochrome c and the integral membrane protein cytochrome c oxidase (COX) is the terminal reaction in the respiratory chain. The first step in this reaction is the diffusional association of cytochrome c toward COX, and it is still not completely clear whether cytochrome c diffuses in the bulk solution while encountering COX, or whether it prefers to diffuse laterally on the membrane surface. This is a rather crucial question, since in the latter case the association would be strongly dependent on the lipid composition and the presence of additional membrane proteins. We applied Brownian dynamics simulations to investigate the effect of an atomistically modeled dipalmitoyl phosphatidylcholine membrane on the association behavior of cytochrome c toward COX from Paracoccus denitrificans. We studied the negatively charged, physiological electron-transfer partner of COX, cytochrome c(552), and the positively charged horse-heart cytochrome c. As expected, both cytochrome c species prefer diffusion in bulk solution while associating toward COX embedded in a membrane, where the partial charges of the lipids were switched off, and the corresponding optimal association pathways largely overlap with the association toward fully solvated COX. Remarkably, after switching on the lipid partial charges, both cytochrome c species were strongly attracted by the inhomogeneous charge distribution caused by the zwitterionic lipid headgroups. This effect is particularly enhanced for horse-heart cytochrome c and is stronger at lower ionic strength. We therefore conclude that in the presence of a polar or even a charged membrane, cytochrome c diffuses laterally rather than in three dimensions. PMID:19254533

  9. Regulation of protein mobility via thermal membrane undulations.

    PubMed

    Brown, Frank L H

    2003-02-01

    The in-plane diffusivelike motion of membrane bound proteins on the surface of cells is considered. We suggest, on the basis of theoretical arguments and simulation, that thermally excited undulations of the lipid bilayer may serve as a mechanism for proteins to hop between adjacent regions on the cell surface separated by barriers composed of internal cellular structure (e.g., the cytoskeleton). We specifically investigate the mobility of band 3 dimer on the surface of red blood cells where the spectrin cytoskeletal meshwork defines a series of "corrals" on the cell surface known to hinder protein motion. Previous models of this system have postulated that the cytoskeleton must deform to allow passage of membrane bound proteins out of these corral regions and have ignored fluctuations of the bilayer. Our model provides a complementary mechanism and we posit that the mobility of real proteins in real cells is likely the result of several mechanisms acting in parallel. PMID:12547768

  10. Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves L-lysine formation.

    PubMed

    Kabus, Armin; Georgi, Tobias; Wendisch, Volker F; Bott, Michael

    2007-05-01

    A critical factor in the biotechnological production of L: -lysine with Corynebacterium glutamicum is the sufficient supply of NADPH. The membrane-integral nicotinamide nucleotide transhydrogenase PntAB of Escherichia coli can use the electrochemical proton gradient across the cytoplasmic membrane to drive the reduction of NADP(+) via the oxidation of NADH. As C. glutamicum does not possess such an enzyme, we expressed the E. coli pntAB genes in the genetically defined C. glutamicum lysine-producing strain DM1730, resulting in membrane-associated transhydrogenase activity of 0.7 U/mg protein. When cultivated in minimal medium with 10% (w/v) carbon source, the presence of transhydrogenase slightly reduced glucose consumption, whereas the consumption of fructose, glucose plus fructose, and, in particular, sucrose was stimulated. Biomass was increased by pntAB expression between 10 and 30% on all carbon sources tested. Most importantly, the lysine concentration was increased in the presence of transhydrogenase by approximately 10% on glucose, approximately 70% on fructose, approximately 50% on glucose plus fructose, and even by approximately 300% on sucrose. Thus, the presence of a proton-coupled transhydrogenase was shown to be an efficient way to improve lysine production by C. glutamicum. In contrast, pntAB expression had a negative effect on growth and glutamate production of C. glutamicum wild type. PMID:17216441

  11. The Membrane-Bound NAC Transcription Factor ANAC013 Functions in Mitochondrial Retrograde Regulation of the Oxidative Stress Response in Arabidopsis[C][W

    PubMed Central

    De Clercq, Inge; Vermeirssen, Vanessa; Van Aken, Olivier; Vandepoele, Klaas; Murcha, Monika W.; Law, Simon R.; Inzé, Annelies; Ng, Sophia; Ivanova, Aneta; Rombaut, Debbie; van de Cotte, Brigitte; Jaspers, Pinja; Van de Peer, Yves; Kangasjärvi, Jaakko; Whelan, James; Van Breusegem, Frank

    2013-01-01

    Upon disturbance of their function by stress, mitochondria can signal to the nucleus to steer the expression of responsive genes. This mitochondria-to-nucleus communication is often referred to as mitochondrial retrograde regulation (MRR). Although reactive oxygen species and calcium are likely candidate signaling molecules for MRR, the protein signaling components in plants remain largely unknown. Through meta-analysis of transcriptome data, we detected a set of genes that are common and robust targets of MRR and used them as a bait to identify its transcriptional regulators. In the upstream regions of these mitochondrial dysfunction stimulon (MDS) genes, we found a cis-regulatory element, the mitochondrial dysfunction motif (MDM), which is necessary and sufficient for gene expression under various mitochondrial perturbation conditions. Yeast one-hybrid analysis and electrophoretic mobility shift assays revealed that the transmembrane domain–containing NO APICAL MERISTEM/ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR/CUP-SHAPED COTYLEDON transcription factors (ANAC013, ANAC016, ANAC017, ANAC053, and ANAC078) bound to the MDM cis-regulatory element. We demonstrate that ANAC013 mediates MRR-induced expression of the MDS genes by direct interaction with the MDM cis-regulatory element and triggers increased oxidative stress tolerance. In conclusion, we characterized ANAC013 as a regulator of MRR upon stress in Arabidopsis thaliana. PMID:24045019

  12. Microbial oxidative sulfur metabolism: biochemical evidence of the membrane-bound heterodisulfide reductase-like complex of the bacterium Aquifex aeolicus.

    PubMed

    Boughanemi, Souhela; Lyonnet, Jordan; Infossi, Pascale; Bauzan, Marielle; Kosta, Artémis; Lignon, Sabrina; Giudici-Orticoni, Marie-Thérèse; Guiral, Marianne

    2016-08-01

    The Hdr (heterodisulfide reductase)-like enzyme is predicted, from gene transcript profiling experiments previously published, to be essential in oxidative sulfur metabolism in a number of bacteria and archaea. Nevertheless, no biochemical and physicochemical data are available so far about this enzyme. Genes coding for it were identified in Aquifex aeolicus, a Gram-negative, hyperthermophilic, chemolithoautotrophic and microaerophilic bacterium that uses inorganic sulfur compounds as electron donor to grow. We provide biochemical evidence that this Hdr-like enzyme is present in this sulfur-oxidizing prokaryote (cultivated with thiosulfate or elemental sulfur). We demonstrate, by immunolocalization and cell fractionation, that Hdr-like enzyme is associated, presumably monotopically, with the membrane fraction. We show by co-immunoprecipitation assay or partial purification, that the Hdr proteins form a stable complex composed of at least five subunits, HdrA, HdrB1, HdrB2, HdrC1 and HdrC2, present in two forms of high molecular mass on native gel (∼240 and 450 kDa). These studies allow us to propose a revised model for dissimilatory sulfur oxidation pathways in A. aeolicus, with Hdr predicted to generate sulfite. PMID:27284018

  13. YM155 sensitizes triple-negative breast cancer to membrane-bound TRAIL through p38 MAPK- and CHOP-mediated DR5 upregulation.

    PubMed

    Pennati, Marzia; Sbarra, Stefania; De Cesare, Michelandrea; Lopergolo, Alessia; Locatelli, Silvia L; Campi, Elisa; Daidone, Maria Grazia; Carlo-Stella, Carmelo; Gianni, Alessandro M; Zaffaroni, Nadia

    2015-01-15

    Because available treatments have limited efficacy in triple-negative breast cancer (TNBC), the identification of new therapeutic strategies to improve patients' outcome is urgently needed. In our study, we investigated the effects of the administration of the small molecule selective survivin suppressant YM155, alone or in association with CD34+ cells transduced with a replication-deficient adenovirus encoding the human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene (CD34-TRAIL+ cells), in three TNBC cell models. YM155 exposure significantly impaired TNBC cell growth and selectively modulated survivin expression at both mRNA and protein level. In addition, co-culturing YM155-treated TNBC cells with CD34-TRAIL+ cells resulted in markedly increased cytotoxic effect and apoptotic response in comparison with single treatments. Such a chemosensitizing effect was observed only in TNBC cells inherently expressing DR5 and relied on the ability of YM155 to upregulate DR5 expression through a p38 MAPK- and CHOP-dependent mechanism. YM155/CD34-TRAIL+ combination also showed a significant inhibitory effect on the growth of DR5-expressing TNBC cells following xenotransplantation into NOD/SCID mice, in the absence of toxicity. Overall, our data (i) provide, for the first time, evidence that YM155 sensitizes TNBC cells to CD34-TRAIL+ cells-induced apoptosis by a mechanism involving the downregulation of survivin and the simultaneous p38 MAPK- and CHOP-mediated upregulation of DR5, and (ii) suggest the combination of YM155 with TRAIL-armed CD34+ progenitor cells as a promising therapeutic option for patients with TNBC expressing DR5. PMID:24866585

  14. Minimal influence of G-protein null mutations on ozone-induced changes in gene expression, foliar injury, gas-exchange and peroxidase activity in Arabidopsis thaliana L

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ozone uptake by plants leads to an increase in reactive oxygen species (ROS) in the intercellular space of leaves and induces signalling processes reported to involve the membrane-bound heterotrimeric G-protein complex. Therefore, potential G-protein-mediated response mechanisms to ozone were compar...

  15. Synthesis of membrane-bound colony-stimulating factor 1 (CSF-1) and downmodulation of CSF-1 receptors in NIH 3T3 cells transformed by cotransfection of the human CSF-1 and c-fms (CSF-1 receptor) genes.

    PubMed Central

    Rettenmier, C W; Roussel, M F; Ashmun, R A; Ralph, P; Price, K; Sherr, C J

    1987-01-01

    NIH 3T3 cells cotransfected with the human c-fms proto-oncogene together with a 1.6-kilobase cDNA clone encoding a 256-amino-acid precursor of the human mononuclear phagocyte colony-stimulating factor CSF-1 (M-CSF) undergo transformation by an autocrine mechanism. The number of CSF-1 receptors on the surface of transformed cells was regulated by ligand-induced receptor degradation and was inversely proportional to the quantity of CSF-1 produced. A tyrosine-to-phenylalanine mutation at position 969 near the receptor carboxyl terminus potentiated its transforming efficiency in cells cotransfected by the CSF-1 gene but did not affect receptor downmodulation. CSF-1 was synthesized as an integral transmembrane glycoprotein that was rapidly dimerized through disulfide bonds. The homodimer was externalized at the cell surface, where it underwent proteolysis to yield the soluble growth factor. Trypsin treatment of viable cells cleaved the plasma membrane form of CSF-1 to molecules of a size indistinguishable from that of the extracellular growth factor, suggesting that trypsinlike proteases regulate the rate of CSF-1 release from transformed cells. The data raise the possibility that this form of membrane-bound CSF-1 might stimulate receptors on adjacent cells through direct cell-cell interactions. Images PMID:3039346

  16. Comparison of the oxime-induced reactivation of rhesus monkey, swine and guinea pig erythrocyte acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity.

    PubMed

    Herkert, Nadja M; Lallement, Guy; Clarençon, Didier; Thiermann, Horst; Worek, Franz

    2009-04-28

    Recently, a dynamically working in vitro model with real-time determination of membrane-bound human acetylcholinesterase (AChE) activity was shown to be a versatile model to investigate oxime-induced reactivation kinetics of organophosphate- (OP) inhibited enzyme. In this assay, AChE was immobilized on particle filters which were perfused with acetylthiocholine, Ellman's reagent and phosphate buffer. Subsequently, AChE activity was continuously analyzed in a flow-through detector. Now, it was an intriguing question whether this model could be used with erythrocyte AChE from other species in order to investigate kinetic interactions in the absence of annoying side reactions. Rhesus monkey, swine and guinea pig erythrocytes were a stable and highly reproducible enzyme source. Then, the model was applied to the reactivation of sarin- and paraoxon-inhibited AChE by obidoxime or HI 6 and it could be shown that the derived reactivation rate constants were in good agreement to previous results obtained from experiments with a static model. Hence, this dynamic model offers the possibility to investigate highly reproducible interactions between AChE, OP and oximes with human and animal AChE. PMID:19428926

  17. One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy

    PubMed Central

    Fricke, Franziska; Beaudouin, Joel; Eils, Roland; Heilemann, Mike

    2015-01-01

    Probing the oligomeric state of abundant molecules, such as membrane proteins in intact cells, is essential, but has not been straightforward. We address this challenge with a simple counting strategy that is capable of reporting the oligomeric state of dense, membrane-bound protein complexes. It is based on single-molecule localization microscopy to super-resolve protein structures in intact cells and basic quantitative evaluation. We validate our method with membrane-bound monomeric CD86 and dimeric cytotoxic T-lymphocyte-associated protein as model proteins and confirm their oligomeric states. We further detect oligomerization of CD80 and vesicular stomatitis virus glycoprotein and propose coexistence of monomers and dimers for CD80 and trimeric assembly of the viral protein at the cell membrane. This approach should prove valuable for researchers striving for reliable molecular counting in cells. PMID:26358640

  18. Comparison of the oxime-induced reactivation of erythrocyte and muscle acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity.

    PubMed

    Eckert, Saskia; Eyer, Peter; Herkert, Nadja; Bumm, Rudolf; Weber, Georg; Thiermann, Horst; Worek, Franz

    2008-02-01

    The purpose of these experiments was to compare oxime-induced reactivation rate constants of acetylcholinesterase from different human tissue sources inhibited by organophosphorus compounds. To this end, preliminary testing was necessary to generate a stable system both for working with erythrocytes and musculature. We established a dynamically working in vitro model with a fixed enzyme source in a bioreactor that was perfused with acetylthiocholine, Ellman's reagent and any agent of interest (e.g. nerve agents, oximes) and analyzed in a common HPLC flow-through detector. The enzyme reactor was composed of a particle filter (Millex-GS, 0.22 microm) containing a thin layer of membrane-bound acetylcholinesterase and was kept at constant temperature in a water bath. At constant flow the height of absorbance was directly proportional to the enzyme activity. To start with, we applied this system to human red cell membranes and then adapted the system to acetylcholinesterase of muscle tissue. Homogenate (Ultra-Turrax and Potter-Elvehjem homogenizer) of human muscle tissue (intercostal musculature) was applied to the same particle filter and perfused in a slightly modified way, as done with human red cell membranes. We detected no decrease of acetylcholinesterase activity within 2.5h and we reproducibly determined reactivation rate constants for reactivation with obidoxime (10 microM) or HI 6 (30 microM) of sarin-inhibited human muscle acetylcholinesterase (0.142+/-0.004 min(-1) and 0.166+/-0.008 min(-1), respectively). The reactivation rate constants of erythrocyte and muscular acetylcholinesterase differed only slightly, highlighting erythrocyte acetylcholinesterase as a proper surrogate marker. PMID:17977518

  19. Palmitoylation of Hedgehog proteins.

    PubMed

    Buglino, John A; Resh, Marilyn D

    2012-01-01

    Hedgehog (Hh) proteins are secreted signaling proteins that contain amide-linked palmitate at the N-terminus and cholesterol at the C-terminus. Palmitoylation of Hh proteins is critical for effective long- and short-range signaling. The palmitoylation reaction occurs during transit of Hh through the secretory pathway, most likely in the lumen of the ER. Attachment of palmitate to Hh proteins is independent of cholesterol modification and autoprocessing and is catalyzed by Hhat (Hedgehog acyltransferase). Hhat is a member of the membrane bound O-acyltransferase (MBOAT) family, a subgroup of multipass membrane proteins that catalyze transfer of fatty acyl groups to lipids and proteins. Several classes of secreted proteins have recently been shown to be substrates for MBOAT acyltransferases, including Hh proteins and Spitz (palmitoylated by Hhat), Wg/Wnt proteins (modified with palmitate and/or palmitoleate by Porcupine) and ghrelin (octanoylated by ghrelin O-acyltransferase). These findings highlight protein fatty acylation as a mechanism that not only influences membrane binding of intracellular proteins but also regulates the signaling range and efficacy of secreted proteins. PMID:22391306

  20. [Vesiculation and change of erythrocyte membrane proteins in storage of preserved blood and erythrocyte mass at 4 degrees C].

    PubMed

    Chernitskiĭ, E A; Fedorovich, I E; Slobozhanina, E I

    1993-01-01

    Erythrocyte vesiculation and changes in membrane protein composition during storage of blood and red cell concentrates in solution "Glugicir" at 4 +/- 2 degrees C have been studied. It is shown that the main part of vesicles is shedding after 9 days of storage. This process corresponds to a decrease of protein 3 and to polyphase changes of membrane-bound hemoglobin as is shown by gel-electrophoresis. During 9-day storage, when erythrocyte ATP is constant and the vesicle shedding is small, an increase in membrane-bound hemoglobin and a decrease of spectrin in isolated membranes take place. The above changes are not the result of oxidative processes in membrane lipid bilayer or in hemoglobin. There was also protein destruction in membranes of stored erythrocytes. It is suggested that in stored blood two type of erythrocyte vesiculation take place: one is due to cell senescence, the other results from metabolic depletion. PMID:8307298

  1. The signal sequence of exported protein-1 directs the green fluorescent protein to the parasitophorous vacuole of transfected malaria parasites.

    PubMed

    Adisa, Akinola; Rug, Melanie; Klonis, Nectarios; Foley, Michael; Cowman, Alan F; Tilley, Leann

    2003-02-21

    The malaria parasite, Plasmodium falciparum, spends part of its life cycle inside the erythrocytes of its human host. In the mature stages of intraerythrocytic growth, the parasite undertakes extensive remodeling of its adopted cellular home by exporting proteins beyond the confines of its own plasma membrane. To examine the signals involved in export of parasite proteins, we have prepared transfected parasites expressing a chimeric protein comprising the N-terminal region of the Plasmodium falciparum exported protein-1 appended to green fluorescent protein. The majority of the population of the chimeric protein appears to be correctly processed and trafficked to the parasitophorous vacuole, indicating that this is the default destination for protein secretion. Some of the protein is redirected to the parasite food vacuole and further degraded. Photobleaching studies reveal that the parasitophorous vacuole contains subcompartments that are only partially interconnected. Dual labeling with the lipid probe, BODIPY-TR-ceramide, reveals the presence of membrane-bound extensions that can bleb from the parasitophorous vacuole to produce double membrane-bound compartments. We also observed regions and extensions of the parasitophorous vacuole, where there is segregation of the lumenal chimera from the lipid components. These regions may represent sites for the sorting of proteins destined for the trafficking to sites beyond the parasitophorous vacuole membrane. PMID:12456681

  2. Curvature Sorting of Peripheral Proteins on Solid-Supported Wavy Membranes

    PubMed Central

    Hsieh, Wan-Ting; Hsu, Chih-Jung; Capraro, Benjamin R.; Wu, Tingting; Chen, Chi-Mon; Yang, Shu; Baumgart, Tobias

    2013-01-01

    Cellular membrane deformation and the associated redistribution of membrane-bound proteins are important aspects of membrane function. Current model membrane approaches for studying curvature sensing are limited to positive curvatures, and often require complex and delicate experimental setups. To overcome these challenges, we fabricated a wavy substrate imposing a range of curvatures onto an adhering lipid bilayer membrane. We examined the curvature sorting of several peripheral proteins binding to the wavy membrane and observed them to partition into distinct regions of curvature. Furthermore, single molecule imaging experiments suggested that curvature sensing of proteins on low-curvature substrates requires cooperative interactions. PMID:22881196

  3. Selection for genes encoding secreted proteins and receptors.

    PubMed Central

    Klein, R D; Gu, Q; Goddard, A; Rosenthal, A

    1996-01-01

    Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms. Despite that, the systematic identification of genes encoding these proteins has not been possible. We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast. Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins. These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels. The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states. Images Fig. 1 PMID:8692953

  4. Regulation of mitochondrial protein import by cytosolic kinases.

    PubMed

    Schmidt, Oliver; Harbauer, Angelika B; Rao, Sanjana; Eyrich, Beate; Zahedi, René P; Stojanovski, Diana; Schönfisch, Birgit; Guiard, Bernard; Sickmann, Albert; Pfanner, Nikolaus; Meisinger, Chris

    2011-01-21

    Mitochondria import a large number of nuclear-encoded proteins via membrane-bound transport machineries; however, little is known about regulation of the preprotein translocases. We report that the main protein entry gate of mitochondria, the translocase of the outer membrane (TOM complex), is phosphorylated by cytosolic kinases-in particular, casein kinase 2 (CK2) and protein kinase A (PKA). CK2 promotes biogenesis of the TOM complex by phosphorylation of two key components, the receptor Tom22 and the import protein Mim1, which in turn are required for import of further Tom proteins. Inactivation of CK2 decreases the levels of the TOM complex and thus mitochondrial protein import. PKA phosphorylates Tom70 under nonrespiring conditions, thereby inhibiting its receptor activity and the import of mitochondrial metabolite carriers. We conclude that cytosolic kinases exert stimulatory and inhibitory effects on biogenesis and function of the TOM complex and thus regulate protein import into mitochondria. PMID:21215441

  5. Proteins interacting with Membranes: Protein Sorting and Membrane Shaping

    NASA Astrophysics Data System (ADS)

    Callan-Jones, Andrew

    2015-03-01

    Membrane-bound transport in cells requires generating membrane curvature. In addition, transport is selective, in order to establish spatial gradients of membrane components in the cell. The mechanisms underlying cell membrane shaping by proteins and the influence of curvature on membrane composition are active areas of study in cell biophysics. In vitro approaches using Giant Unilamellar Vesicles (GUVs) are a useful tool to identify the physical mechanisms that drive sorting of membrane components and membrane shape change by proteins. I will present recent work on the curvature sensing and generation of IRSp53, a protein belonging to the BAR family, whose members, sharing a banana-shaped backbone, are involved in endocytosis. Pulling membrane tubes with 10-100 nm radii from GUVs containing encapsulated IRSp53 have, unexpectedly, revealed a non-monotonic dependence of the protein concentration on the tube as a function of curvature. Experiments also show that bound proteins alter the tube mechanics and that protein phase separation along the tube occurs at low tensions. I will present accompanying theoretical work that can explain these findings based on the competition between the protein's intrinsic curvature and the effective rigidity of a membrane-protein patch.

  6. Interaction of the Intermembrane Space Domain of Tim23 Protein with Mitochondrial Membranes*

    PubMed Central

    Bajaj, Rakhi; Munari, Francesca; Becker, Stefan; Zweckstetter, Markus

    2014-01-01

    Tim23 mediates protein translocation into mitochondria. Although inserted into the inner membrane, the dynamic association of its intermembrane space (IMS) domain with the outer membrane promotes protein import. However, little is known about the molecular basis of this interaction. Here, we demonstrate that the IMS domain of Tim23 tightly associates with both inner and outer mitochondrial membrane-like membranes through a hydrophobic anchor at its N terminus. The structure of membrane-bound Tim23IMS is highly dynamic, allowing recognition of both the incoming presequence and other translocase components at the translocation contact. Cardiolipin enhances Tim23 membrane attachment, suggesting that cardiolipin can influence preprotein import. PMID:25349212

  7. Laser flash photolysis as a probe of redox protein-membrane interactions: Effect of binding of spinach plastocyanin and horse cytochrome c to lipid bilayer vesicles on the kinetics of reduction by flavin semiquinone

    SciTech Connect

    Senthilathipan, V.; Tollin, G. )

    1989-02-07

    Spinach plastocyanin binds to both electrically neutral and positively charged lipid bilayer vesicles whereas cytochrome c only binds electrostatically to negatively charged vesicles. Laser flash photolysis using lumiflavin semiquinone as a reductant demonstrates that the reactivity of plastocyanin is increased as much as 6-fold when it is membrane bound whereas the rate constant for cytochrome c reduction is decreased by approximately a factor of 3. Membrane-bound plastocyanin reduction occurs via a two-step mechanism, probably involving prior association of lumiflavin semiquinone with the bilayer. In contrast, cytochrome c reduction in the membrane-bound state follows simple second-order kinetics, implying that the redox site in the bound state is still accessible to lumiflavin semiquinone in solution, although the rate constant is decreased by approximately 3-fold. These results are interpreted as indicating that the bilayer-protein interaction with plastocyanin leads to a steric blockage of the electron-transfer site from the aqueous phase. Little or no hindrance of the redox site occurs with cytochrome c, suggesting a high degree of mobility of this protein on the bilayer surface. Although the increase in plastocyanin reactivity upon binding to the bilayer is quite interesting, its cause remains unclear and requires further study. The results illustrate the utility of laser flash photolysis as a probe of membrane-protein interactions.

  8. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  9. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  10. Thylakoid-Bound FtsH Proteins Facilitate Proper Biosynthesis of Photosystem I1[OPEN

    PubMed Central

    2016-01-01

    Thylakoid membrane-bound FtsH proteases have a well-characterized role in degradation of the photosystem II (PSII) reaction center protein D1 upon repair of photodamaged PSII. Here, we show that the Arabidopsis (Arabidopsis thaliana) var1 and var2 mutants, devoid of the FtsH5 and FtsH2 proteins, respectively, are capable of normal D1 protein turnover under moderate growth light intensity. Instead, they both demonstrate a significant scarcity of PSI complexes. It is further shown that the reduced level of PSI does not result from accelerated photodamage of the PSI centers in var1 or var2 under moderate growth light intensity. On the contrary, radiolabeling experiments revealed impaired synthesis of the PsaA/B reaction center proteins of PSI, which was accompanied by the accumulation of PSI-specific assembly factors. psaA/B transcript accumulation and translation initiation, however, occurred in var1 and var2 mutants as in wild-type Arabidopsis, suggesting problems in later stages of PsaA/B protein expression in the two var mutants. Presumably, the thylakoid membrane-bound FtsH5 and FtsH2 have dual functions in the maintenance of photosynthetic complexes. In addition to their function as a protease in the degradation of the photodamaged D1 protein, they also are required, either directly or indirectly, for early assembly of the PSI complexes. PMID:27208291

  11. Platelet morphology and membrane bound calcium in porcine stress syndrome.

    PubMed

    Basrur, P K; Frombach, S; McDonell, W N

    1983-01-01

    Platelets of normal and stress susceptible pigs were subjected to electron microscopic examination and energy dispersive x-ray microanalysis in an attempt to study whether the cell membrane defect implicated in porcine stress syndrome is detectable at the platelet level. Electron microscopic studies revealed that the platelets of stress susceptible pigs are morphologically distinguishable from those of normal pigs by virtue of their highly dilated surface connected open canalicular system rendering the "Swiss cheese" appearance and the absence of the circumferential band of microtubules. A comparison of the data from spot analysis of cell membrane and the membranes of the open canalicular system using the energy dispersive microanalysis system showed that the calcium content of the plasma membrane of stress susceptible pigs is significantly lower than that of normal pigs. It would appear that the morphologic feature and the reduced levels of calcium bound to platelet membrane are related to the generalized membrane defect postulated to be the primary lesion in porcine stress syndrome and that these parameters could be included among the criteria for the detection of stress susceptible pigs. PMID:6635547

  12. Are soluble and membrane-bound rat brain acetylcholinesterase different

    SciTech Connect

    Andres, C.; el Mourabit, M.; Stutz, C.; Mark, J.; Waksman, A. )

    1990-11-01

    Salt-soluble and detergent-soluble acetylcholinesterases (AChE) from adult rat brain were purified to homogeneity and studied with the aim to establish the differences existing between these two forms. It was found that the enzymatic activities of the purified salt-soluble AChE as well as the detergent-soluble AChE were dependent on the Triton X-100 concentration. Moreover, the interaction of salt-soluble AChE with liposomes suggests amphiphilic behaviour of this enzyme. Serum cholinesterase (ChE) did not bind to liposomes but its activity was also detergent-dependent. Detergent-soluble AChE remained in solution below critical micellar concentrations of Triton X-100. SDS polyacrylamide gel electrophoresis of purified, Biobeads-treated and iodinated detergent-soluble 11 S AChE showed, under non reducing conditions, bands of 69 kD, 130 kD and greater than 250 kD corresponding, respectively, to monomers, dimers and probably tetramers of the same polypeptide chain. Under reducing conditions, only a 69 kD band was detected. It is proposed that an amphiphilic environment stabilizes the salt-soluble forms of AChE in the brain in vivo and that detergent-soluble Biobeads-treated 11 S AChE possess hydrophobic domain(s) different from the 20 kD peptide already described.

  13. Calcium Modulation of Plant Plasma Membrane-Bound Atpase Activities

    NASA Technical Reports Server (NTRS)

    Caldwell, C.

    1983-01-01

    The kinetic properties of barley enzyme are discussed and compared with those of other plants. Possibilities for calcium transport in the plasma membrane by proton pump and ATPase-dependent calcium pumps are explored. Topics covered include the ph phase of the enzyme; high affinity of barley for calcium; temperature dependence, activation enthalpy, and the types of ATPase catalytic sites. Attention is given to lipids which are both screened and bound by calcium. Studies show that barley has a calmodulin activated ATPase that is found in the presence of magnesium and calcium.

  14. Transient domain formation in membrane-bound organelles undergoing maturation

    NASA Astrophysics Data System (ADS)

    Dmitrieff, Serge; Sens, Pierre

    2013-12-01

    The membrane components of cellular organelles have been shown to segregate into domains as the result of biochemical maturation. We propose that the dynamical competition between maturation and lateral segregation of membrane components regulates domain formation. We study a two-component fluid membrane in which enzymatic reaction irreversibly converts one component into another and phase separation triggers the formation of transient membrane domains. The maximum domain size is shown to depend on the maturation rate as a power law similar to the one observed for domain growth with time in the absence of maturation, despite this time dependence not being verified in the case of irreversible maturation. This control of domain size by enzymatic activity could play a critical role in regulating exchange between organelles or within compartmentalized organelles such as the Golgi apparatus.

  15. Targeting membrane proteins for antibody discovery using phage display

    PubMed Central

    Jones, Martina L.; Alfaleh, Mohamed A.; Kumble, Sumukh; Zhang, Shuo; Osborne, Geoffrey W.; Yeh, Michael; Arora, Neetika; Hou, Jeff Jia Cheng; Howard, Christopher B.; Chin, David Y.; Mahler, Stephen M.

    2016-01-01

    A critical factor in the successful isolation of new antibodies by phage display is the presentation of a correctly folded antigen. While this is relatively simple for soluble proteins which can be purified and immobilized onto a plastic surface, membrane proteins offer significant challenges for antibody discovery. Whole cell panning allows presentation of the membrane protein in its native conformation, but is complicated by a low target antigen density, high background of irrelevant antigens and non-specific binding of phage particles to cell surfaces. The method described here uses transient transfection of alternating host cell lines and stringent washing steps to address each of these limitations. The successful isolation of antibodies from a naive scFv library is described for three membrane bound proteins; human CD83, canine CD117 and bat CD11b. PMID:27189586

  16. Targeting membrane proteins for antibody discovery using phage display.

    PubMed

    Jones, Martina L; Alfaleh, Mohamed A; Kumble, Sumukh; Zhang, Shuo; Osborne, Geoffrey W; Yeh, Michael; Arora, Neetika; Hou, Jeff Jia Cheng; Howard, Christopher B; Chin, David Y; Mahler, Stephen M

    2016-01-01

    A critical factor in the successful isolation of new antibodies by phage display is the presentation of a correctly folded antigen. While this is relatively simple for soluble proteins which can be purified and immobilized onto a plastic surface, membrane proteins offer significant challenges for antibody discovery. Whole cell panning allows presentation of the membrane protein in its native conformation, but is complicated by a low target antigen density, high background of irrelevant antigens and non-specific binding of phage particles to cell surfaces. The method described here uses transient transfection of alternating host cell lines and stringent washing steps to address each of these limitations. The successful isolation of antibodies from a naive scFv library is described for three membrane bound proteins; human CD83, canine CD117 and bat CD11b. PMID:27189586

  17. Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells

    PubMed Central

    Baghirova, Sabina; Hughes, Bryan G.; Hendzel, Michael J.; Schulz, Richard

    2015-01-01

    Many types of studies require the localization of a protein to, or isolation of enriched protein from a specific cellular compartment. Many protocols in the literature and from commercially available kits claim to yield pure cellular fractions. However, in our hands, the former often do not work effectively and the latter may be prohibitively expensive if a large number of fractionations are required. Furthermore, the largely proprietary composition of reagents in commercial kits means that the user is not able to make adjustments if, for example, a particular component affects the activity of a protein of interest. The method described here allows the isolation of purified proteins from three cellular fractions: the cytosol, membrane-bound organelles, and the nucleus. It uses gentle buffers with increasing detergent strength that sequentially lyse the cell membrane, organelle membranes and finally the nuclear membrane.•Quick, simple to replicate or adjust; this method does not require expensive reagents or use of commercial kits•The protocol can be applied to tissue samples or cultured cells without changing buffer components•Yields purified fractions of cytosolic, membrane bound and nuclear proteins, with the proper distribution of the appropriate subcellular markers: GAPDH, VDAC, SERCA2 and lamin A/C PMID:26740924

  18. Towards understanding of Nipah virus attachment protein assembly and the role of protein affinity and crowding for membrane curvature events.

    SciTech Connect

    Stachowiak, Jeanne C.; Hayden, Carl C.; Negrete, Oscar A.; Davis, Ryan Wesley; Sasaki, Darryl Yoshio

    2013-10-01

    Pathogenic viruses are a primary threat to our national security and to the health and economy of our world. Effective defense strategies to combat viral infection and spread require the development of understanding of the mechanisms that these pathogens use to invade the host cell. We present in this report results of our research into viral particle recognition and fusion to cell membranes and the role that protein affinity and confinement in lipid domains plays in membrane curvature in cellular fusion and fission events. Herein, we describe 1) the assembly of the G attachment protein of Nipah virus using point mutation studies to define its role in viral particle fusion to the cell membrane, 2) how lateral pressure of membrane bound proteins induce curvature in model membrane systems, and 3) the role of membrane curvature in the selective partitioning of molecular receptors and specific affinity of associated proteins.

  19. Membrane association of sucrose synthase: changes during the graviresponse and possible control by protein phosphorylation

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1997-01-01

    Sucrose synthase (SuSy) plays an important role in sucrose degradation and occurs both as a soluble and as a membrane-associated enzyme in higher plants. We show that membrane association can vary in vivo in response to gravistimulation, apparently involving SuSy dephosphorylation, and is a reversible process in vitro. Phosphorylation of SuSy has little effect on its activity but decreases its surface hydrophobicity as reported with the fluorescent probe bis-ANS. We postulate that phosphorylation of SuSy (and perhaps other membrane proteins) is involved in the release of the membrane-bound enzyme in part as a result of decreased surface hydrophobicity.

  20. Regulation of innate immune signalling pathways by the tripartite motif (TRIM) family proteins

    PubMed Central

    Kawai, Taro; Akira, Shizuo

    2011-01-01

    The innate immune system recognizes microbial components through pattern-recognition receptors (PRRs), including membrane-bound Toll-like receptors and cytosolic receptors such as RIG-I-like receptors and deoxyribonucleic acid (DNA) sensors. These PRRs trigger distinct signal transduction pathways that culminate in induction of an array of cytokines and other mediators required for host defense. The tripartite motif (TRIM) family is a diverse family of RING finger domain-containing proteins, which are involved in a variety of cellular functions. Importantly, recent studies have shown that they are also involved in the regulation of innate immune responses through the modulation of PRR signalling pathways. PMID:21826793

  1. Intrinsic membrane association of Drosophila cysteine string proteins.

    PubMed

    Mastrogiacomo, A; Kohan, S A; Whitelegge, J P; Gundersen, C B

    1998-09-25

    Cysteine string proteins (csps) are highly conserved constituents of vertebrate and invertebrate secretory organelles. Biochemical and immunoprecipitation experiments implied that vertebrate csps were integral membrane proteins that were tethered to the outer leaflet of secretory vesicles via the fatty acyl residues of their extensively acylated cysteine string. Independently, work of others suggested that Drosophila csps were peripheral membrane proteins that were anchored to membranes by a mechanism that was independent of the cysteine string and its fatty acyl residues. We extended these investigation and found first that sodium carbonate treatment partially stripped both csps and the integral membrane protein, synaptotagmin, from Drosophila membranes. Concomitantly, carbonate released fatty acids into the medium, arguing that it has a mild, solubilizing effect on these membranes. Second, we observed that Drosophila csps behaved like integral membrane proteins in Triton X-114 partitioning experiments. Third, we found that when membrane-bound csps were deacylated, they remained membrane bound. Moreover, it appeared that hydrophobic interactions were necessary for this persistent membrane association of csps. Thus, neither reducing conditions, urea, nor chaotropic agents displaced deacylated csps from membranes. Only detergents were effective in solubilizing deacylated csps. Finally, by virtue of the inaccessibility of deacylated csps to thiol alkylation by the membrane-impermeant alkylating reagent, iodoacetic acid, we inferred that it was the cysteine string domain that mediated the membrane association of deacylated csps. Thus, we conclude that under physiological conditions csps are integral membrane proteins of secretory organelles, and that the cysteine string domain plays a vital role in the membrane association of these proteins. PMID:9771899

  2. Protein-driven membrane stresses in fusion and fission

    PubMed Central

    Kozlov, Michael M.; McMahon, Harvey T.; Chernomordik, Leonid V.

    2013-01-01

    Cellular membranes undergo continuous remodeling. Exocytosis and endocytosis, mitochondrial fusion and fission, entry of enveloped viruses into host cellsand release of the newly assembled virions, cell-to-cell fusion and cell division, and budding and fusion of transport carriers all proceed via topologically similar, but oppositely ordered, membrane rearrangements. The biophysical similarities and differences between membrane fusion and fission become more evident if we disregard the accompanying biological processes and consider only remodeling of the lipid bilayer. The forces that determine the bilayer propensity to undergo fusion or fission come from proteins and inmost cases from membrane-bound proteins. In this review, we consider the mechanistic principles underlying the fusion and fission reactions and discuss the current hypotheses on how specific proteins act in the two types of membrane remodeling. PMID:20638285

  3. Shock wave induced damage of a protein by void collapse

    DOE PAGESBeta

    Lau, Edmond Y.; Berkowitz, Max L.; Schwegler, Eric R.

    2016-01-05

    In this study, we report on a series of molecular dynamics simulations that were used to examine the effects of shockwaves on a membrane bound ion channel. A planar shockwave was found to compress the ion channel upon impact but the protein geometry resembles the initial structure as soon as the solvent density begins to dissipate. When a void was placed in close proximity to the membrane, the shockwave proved to be much more destructive to the protein due to formation of a nanojet that results from the asymmetric collapse of the void. The nanojet was able to cause significantmore » structural changes to the protein even at low particle velocities that are not able to directly cause poration of the membrane.« less

  4. The alternative oxidases: simple oxidoreductase proteins with complex functions.

    PubMed

    Young, Luke; Shiba, Tomoo; Harada, Shigeharu; Kita, Kiyoshi; Albury, Mary S; Moore, Anthony L

    2013-10-01

    The alternative oxidases are membrane-bound monotopic terminal electron transport proteins found in all plants and in some agrochemically important fungi and parasites including Trypansoma brucei, which is the causative agent of trypanosomiasis. They are integral membrane proteins and reduce oxygen to water in a four electron process. The recent elucidation of the crystal structure of the trypanosomal alternative oxidase at 2.85 Å (1 Å=0.1 nm) has revealed salient structural features necessary for its function. In the present review we compare the primary and secondary ligation spheres of the alternative oxidases with other di-iron carboxylate proteins and propose a mechanism for the reduction of oxygen to water. PMID:24059524

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

    PubMed

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

    1998-01-01

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

  6. The 43-K protein, v1, associated with acetylcholine receptor containing membrane fragments is an actin-binding protein.

    PubMed Central

    Walker, J H; Boustead, C M; Witzemann, V

    1984-01-01

    Acetylcholine receptor enriched membrane fragments were obtained from the electric organs of Torpedo marmorata. The purified membrane fragments contained several proteins in addition to the acetylcholine receptor subunits. One of these was shown to be actin by means of immune blotting with a monoclonal antibody. Brief treatment of the membranes with pH 11.0 buffer removed actin and the other non-receptor proteins including the receptor-associated 43 000 mol. wt. polypeptide. This polypeptide was shown to bind actin after transferring the proteins from one- and two-dimensional polyacrylamide gels to nitrocellulose paper and incubating the nitrocellulose blots with actin. Specifically bound actin was demonstrated using the monoclonal antibodies to actin. No calcium or calmodulin dependency of binding was observed. The findings suggest that the 43 000 mol. wt. polypeptide is a link between the membrane-bound acetylcholine receptor and the cytoskeleton. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6389118

  7. Return to sender: use of Plasmodium ER retrieval sequences to study protein transport in the infected erythrocyte and predict putative ER protein families.

    PubMed

    Külzer, Simone; Gehde, Nina; Przyborski, Jude M

    2009-06-01

    We have investigated how knowledge of endoplasmic reticulum (ER) retrieval signals can be used to study specific trafficking pathways in the malaria-infected erythrocyte. We show that addition of various lumenal ER retrieval signals to soluble green fluorescent protein (GFP) chimaera causes retrieval of the fusion protein in the parasite's ER. In contrast, adding these signals to the C-terminus of a membrane bound protein does not affect its eventual sub-cellular localization. This demonstrates proof of principle that ER retrieval signals can be used to study the solubility state of Plasmodium falciparum proteins during their transport to the host erythrocyte. Furthermore, using our knowledge of ER retrieval signals, we identify Plasmodium ER protein families and assign putative functions to them. PMID:19294420

  8. The VPS33B-binding protein VPS16B is required in megakaryocyte and platelet α-granule biogenesis

    PubMed Central

    Urban, Denisa; Li, Ling; Christensen, Hilary; Pluthero, Fred G.; Chen, Shao Zun; Puhacz, Michael; Garg, Parvesh M.; Lanka, Kiran K.; Cummings, James J.; Kramer, Helmut; Wasmuth, James D.; Parkinson, John

    2012-01-01

    Patients with platelet α or dense δ-granule defects have bleeding problems. Although several proteins are known to be required for δ-granule development, less is known about α-granule biogenesis. Our previous work showed that the BEACH protein NBEAL2 and the Sec1/Munc18 protein VPS33B are required for α-granule biogenesis. Using a yeast two-hybrid screen, mass spectrometry, coimmunoprecipitation, and bioinformatics studies, we identified VPS16B as a VPS33B-binding protein. Immunoblotting confirmed VPS16B expression in various human tissues and cells including megakaryocytes and platelets, and also in megakaryocytic Dami cells. Characterization of platelets from a patient with arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome containing mutations in C14orf133 encoding VPS16B revealed pale-appearing platelets in blood films and electron microscopy revealed a complete absence of α-granules, whereas δ-granules were observed. Soluble and membrane-bound α-granule proteins were reduced or undetectable, suggesting that both releasable and membrane-bound α-granule constituents were absent. Immunofluorescence microscopy of Dami cells stably expressing GFP-VPS16B revealed that similar to VPS33B, GFP-VPS16B colocalized with markers of the trans-Golgi network, late endosomes and α-granules. We conclude that VPS16B, similar to its binding partner VPS33B, is essential for megakaryocyte and platelet α-granule biogenesis. PMID:23002115

  9. The VPS33B-binding protein VPS16B is required in megakaryocyte and platelet α-granule biogenesis.

    PubMed

    Urban, Denisa; Li, Ling; Christensen, Hilary; Pluthero, Fred G; Chen, Shao Zun; Puhacz, Michael; Garg, Parvesh M; Lanka, Kiran K; Cummings, James J; Kramer, Helmut; Wasmuth, James D; Parkinson, John; Kahr, Walter H A

    2012-12-13

    Patients with platelet α or dense δ-granule defects have bleeding problems. Although several proteins are known to be required for δ-granule development, less is known about α-granule biogenesis. Our previous work showed that the BEACH protein NBEAL2 and the Sec1/Munc18 protein VPS33B are required for α-granule biogenesis. Using a yeast two-hybrid screen, mass spectrometry, coimmunoprecipitation, and bioinformatics studies, we identified VPS16B as a VPS33B-binding protein. Immunoblotting confirmed VPS16B expression in various human tissues and cells including megakaryocytes and platelets, and also in megakaryocytic Dami cells. Characterization of platelets from a patient with arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome containing mutations in C14orf133 encoding VPS16B revealed pale-appearing platelets in blood films and electron microscopy revealed a complete absence of α-granules, whereas δ-granules were observed. Soluble and membrane-bound α-granule proteins were reduced or undetectable, suggesting that both releasable and membrane-bound α-granule constituents were absent. Immunofluorescence microscopy of Dami cells stably expressing GFP-VPS16B revealed that similar to VPS33B, GFP-VPS16B colocalized with markers of the trans-Golgi network, late endosomes and α-granules. We conclude that VPS16B, similar to its binding partner VPS33B, is essential for megakaryocyte and platelet α-granule biogenesis. PMID:23002115

  10. Mitsugumin 56 (hedgehog acyltransferase-like) is a sarcoplasmic reticulum-resident protein essential for postnatal muscle maturation.

    PubMed

    Van, Bo; Nishi, Miyuki; Komazaki, Shinji; Ichimura, Atsuhiko; Kakizawa, Sho; Nakanaga, Keita; Aoki, Junken; Park, Ki-Ho; Ma, Jianjie; Ueyama, Tomomi; Ogata, Takehiro; Maruyama, Naoki; Takeshima, Hiroshi

    2015-04-28

    Mitsugumin 56 (MG56), also known as the membrane-bound O-acyl-transferase family member hedgehog acyltransferase-like, was identified as a new sarcoplasmic reticulum component in striated muscle. Mg56-knockout mice grew normally for a week after birth, but shortly thereafter exhibited a suckling defect and died under starvation conditions. In the knockout skeletal muscle, regular contractile features were largely preserved, but sarcoplasmic reticulum elements swelled and further developed enormous vacuoles. In parallel, the unfolded protein response was severely activated in the knockout muscle, and presumably disrupted muscle development leading to the suckling failure. Therefore, MG56 seems essential for postnatal skeletal muscle maturation. PMID:25841338

  11. Metabolism of 18S rRNA in rat liver cells in different functional states of protein-synthesizing apparatus

    SciTech Connect

    Chirkov, G.P.; Druzhinina, M.K.; Todorov, I.N.

    1986-04-10

    The ratio of the absolute radioactivities of 28S and 18S RNAs in the fractions of membrane-bound and free polysomes and the fraction of free rat liver ribosomes was studied under conditions of inhibition of translation by cycloheximide, insulin, and cAMP. It was found that insulin and cAMP, in contrast to cycloheximide, do not induce selective degradation of 18S rRNA. The results are discussed from the standpoint of the possible role of the phosphorylation of protein S6 in the degradation of the 40S ribosomal subunit.

  12. Protein characterization of Babesia equi piroplasms isolated from infected horse erythrocytes.

    PubMed

    Ali, S; Sugimoto, C; Matsuda, M; Sugiura, T; Kanemaru, T; Onuma, M; Kamada, M

    1993-01-01

    Proteins of Babesia equi piroplasms were characterized. The piroplasms of B. equi were purified by lysis of infected horse erythrocytes with N2 gas cavitation followed by separation in Percoll density-gradient centrifugation. The relative molecular weights (Mr) of major proteins separated by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 18, 28, 30, 41, 43, 54, 66.5, and 96 kDa. Immunoblot analysis using serum from an experimentally infected horse revealed six immunodominant proteins of 15, 18, 28, 30, 41, and 96 kDa. Two immunodominant proteins of 18 and 28 kDa were membrane-bound proteins as revealed by Triton X-114 phase partitioning. PMID:8295900

  13. Tonoplast-Bound Protein Kinase Phosphorylates Tonoplast Intrinsic Protein 1

    PubMed Central

    Johnson, Kenneth D.; Chrispeels, Maarten J.

    1992-01-01

    Tonoplast intrinsic protein (TIP) is a member of a family of putative membrane channels found in bacteria, animals, and plants. Plants have seed-specific, vegetative/reproductive organ-specific, and water-stress-induced forms of TIP. Here, we report that the seed-specific TIP is a phosphoprotein whose phosphorylation can be monitored in vivo by allowing bean cotyledons to take up [32P]orthophosphate and in vitro by incubating purified tonoplasts with γ-labeled [32P]ATP. Characterization of the in vitro phosphorylation of TIP indicates that a membrane-bound protein kinase phosphorylates TIP in a Ca2+-dependent manner. The capacity of the isolated tonoplast membranes to phosphorylate TIP declined markedly during seed germination, and this decline occurred well before the development-mediated decrease in TIP occurs. Phosphoamino acid analysis of purified, radiolabeled TIP showed that serine is the major, if not only, phosphorylated residue, and cyanogen bromide cleavage yielded a single radioactive peptide peak on a reverse-phase high-performance liquid chromatogram. Estimation of the molecular mass of the cyanogen bromide phosphopeptide by laser desorption mass spectroscopy led to its identification as the hydrophilic N-terminal domain of TIP. The putative phosphate-accepting serine residue occurs in a consensus phosphorylation site for serine/threonine protein kinases. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:16653198

  14. RPE cell surface proteins in normal and dystrophic rats

    SciTech Connect

    Clark, V.M.; Hall, M.O.

    1986-02-01

    Membrane-bound proteins in plasma membrane enriched fractions from cultured rat RPE were analyzed by two-dimensional gel electrophoresis. Membrane proteins were characterized on three increasingly specific levels. Total protein was visualized by silver staining. A maximum of 102 separate proteins were counted in silver-stained gels. Glycoproteins were labeled with 3H-glucosamine or 3H-fucose and detected by autoradiography. Thirty-eight fucose-labeled and 61-71 glucosamine-labeled proteins were identified. All of the fucose-labeled proteins were labeled with glucosamine-derived radioactivity. Proteins exposed at the cell surface were labeled by lactoperoxidase-catalyzed radioiodination prior to preparation of membranes for two-dimensional analysis. Forty separate 125I-labeled surface proteins were resolved by two-dimensional electrophoresis/autoradiography. Comparison with the glycoprotein map showed that a number of these surface labeled proteins were glycoproteins. Two-dimensional maps of total protein, fucose-labeled, and glucosamine-labeled glycoproteins, and 125I-labeled surface proteins of membranes from dystrophic (RCS rdy-p+) and normal (Long Evans or RCS rdy+p+) RPE were compared. No differences in the total protein or surface-labeled proteins were observed. However, the results suggest that a 183K glycoprotein is more heavily glycosylated with glucosamine and fucose in normal RPE membranes as compared to membranes from dystrophic RPE.

  15. The artificial synapse chip: From proteins to prostheses

    NASA Astrophysics Data System (ADS)

    Peterman, Mark Charles

    Most retinal prostheses use an electric field to stimulate retinal circuitry, yet information transfer in the retina is primarily through neurotransmitters. To address this difference, this thesis describes a proof of concept retinal interface based on localized chemical delivery. This system, the Artificial Synapse Chip, is based on a 5 mum aperture in a silicon nitride membrane overlying a microfluidic channel. The effectiveness of this interface is demonstrated by ejecting bradykinin on cultured excitable cells. Even with manual fluidic control, the relationship between the extent of stimulation and concentration is linear, providing enough control to limit stimulation to individual cells. A neurotransmitter-based prosthesis will require advanced fluidic control. This thesis reports the use of electroosmosis to eject or withdraw fluid from an aperture in a channel wall. This effect is demonstrated experimentally, and numerically, using a finite-element method. Our primary device is a prototype interface with four individually addressable apertures in a 2 x 2 array. Using this array, we demonstrate stimulation of both PC12 and retinal ganglion cells. This demonstration of localized chemical stimulation of excitable cells illustrates the potential of this technology for retinal prostheses. As a final application of the Artificial Synapse Chip, we applied the concept to lipid bilayer membranes and membrane-bound proteins. Not only are membrane-bound proteins crucial to the function of biological synapses, but also are important from a technological point of view. In this thesis, we use a Langmuir-Blodgett technique to producing lipid bilayers across apertures in a modified version of the Artificial Synapse Chip. These bilayers display many of the same properties as bilayers across apertures in Teflon films. In addition, these bilayers remain unbroken at transmembrane potentials over +/-400 mV, higher than Teflon-supported bilayers. We also demonstrate single

  16. Redox Regulation of Protein Kinases

    PubMed Central

    Truong, Thu H.; Carroll, Kate S.

    2015-01-01

    Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous H2O2 by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiologic and pathological H2O2 responses. PMID:23639002

  17. Protein-protein, protein-RNA and protein-lipid interactions of signal-recognition particle components in the hyperthermoacidophilic archaeon Acidianus ambivalens.

    PubMed Central

    Moll, Ralf G

    2003-01-01

    The signal-recognition particle (SRP) of one of the most acidophilic and hyperthermophilic archaeal cells, Acidianus ambivalens, and its putative receptor component, FtsY (prokaryotic SRP receptor), were investigated in detail. A. ambivalens Ffh (fifty-four-homologous protein) was shown to be a soluble protein with strong affinity to membranes. In its membrane-residing form, Ffh was extracted from plasma membranes with chaotropic agents like urea, but not with agents diminishing electrostatic interactions. Using unilamellar tetraether phospholipid vesicles, both Ffh and FtsY associate independently from each other in the absence of other factors, suggesting an equilibrium of soluble and membrane-bound protein forms under in vivo conditions. The Ffh protein precipitated from cytosolic cell supernatants with anti-Ffh antibodies, together with an 7 S-alike SRP-RNA, suggesting a stable core ribonucleoprotein composed of both components under native conditions. The SRP RNA of A. ambivalens depicted a size of about 309 nucleotides like the SRP RNA of the related organism Sulfolobus acidocaldarius. A stable heterodimeric complex composed of Ffh and FtsY was absent in cytosolic supernatants, indicating a transiently formed complex during archaeal SRP targeting. The FtsY protein precipitated in cytosolic supernatants with anti-FtsY antisera as a homomeric protein lacking accessory protein components. However, under in vitro conditions, recombinantly generated Ffh and FtsY associate in a nucleotide-independent manner, supporting a structural receptor model with two interacting apoproteins. PMID:12775213

  18. Copper and the Prion Protein: Methods, Structures, Function, and Disease

    NASA Astrophysics Data System (ADS)

    Millhauser, Glenn L.

    2007-05-01

    The transmissible spongiform encephalopathies (TSEs) arise from conversion of the membrane-bound prion protein from PrPC to PrPSc. Examples of the TSEs include mad cow disease, chronic wasting disease in deer and elk, scrapie in goats and sheep, and kuru and Creutzfeldt-Jakob disease in humans. Although the precise function of PrPC in healthy tissues is not known, recent research demonstrates that it binds Cu(II) in an unusual and highly conserved region of the protein termed the octarepeat domain. This review describes recent connections between copper and PrPC, with an emphasis on the electron paramagnetic resonance elucidation of the specific copper-binding sites, insights into PrPC function, and emerging connections between copper and prion disease.

  19. Copper and the prion protein: methods, structures, function, and disease.

    PubMed

    Millhauser, Glenn L

    2007-01-01

    The transmissible spongiform encephalopathies (TSEs) arise from conversion of the membrane-bound prion protein from PrP(C) to PrP(Sc). Examples of the TSEs include mad cow disease, chronic wasting disease in deer and elk, scrapie in goats and sheep, and kuru and Creutzfeldt-Jakob disease in humans. Although the precise function of PrP(C) in healthy tissues is not known, recent research demonstrates that it binds Cu(II) in an unusual and highly conserved region of the protein termed the octarepeat domain. This review describes recent connections between copper and PrP(C), with an emphasis on the electron paramagnetic resonance elucidation of the specific copper-binding sites, insights into PrP(C) function, and emerging connections between copper and prion disease. PMID:17076634

  20. A Usual G-Protein-Coupled Receptor in Unusual Membranes.

    PubMed

    Chawla, Udeep; Jiang, Yunjiang; Zheng, Wan; Kuang, Liangju; Perera, Suchithranga M D C; Pitman, Michael C; Brown, Michael F; Liang, Hongjun

    2016-01-11

    G-protein-coupled receptors (GPCRs) are the largest family of membrane-bound receptors and constitute about 50% of all known drug targets. They offer great potential for membrane protein nanotechnologies. We report here a charge-interaction-directed reconstitution mechanism that induces spontaneous insertion of bovine rhodopsin, the eukaryotic GPCR, into both lipid- and polymer-based artificial membranes. We reveal a new allosteric mode of rhodopsin activation incurred by the non-biological membranes: the cationic membrane drives a transition from the inactive MI to the activated MII state in the absence of high [H(+)] or negative spontaneous curvature. We attribute this activation to the attractive charge interaction between the membrane surface and the deprotonated Glu134 residue of the rhodopsin-conserved ERY sequence motif that helps break the cytoplasmic "ionic lock". This study unveils a novel design concept of non-biological membranes to reconstitute and harness GPCR functions in synthetic systems. PMID:26633591

  1. G-protein-coupled receptors for neurotransmitter amino acids: C-terminal tails, crowded signalosomes.

    PubMed Central

    El Far, Oussama; Betz, Heinrich

    2002-01-01

    G-protein-coupled receptors (GPCRs) represent a superfamily of highly diverse integral membrane proteins that transduce external signals to different subcellular compartments, including nuclei, via trimeric G-proteins. By differential activation of diffusible G(alpha) and membrane-bound G(beta)gamma subunits, GPCRs might act on both cytoplasmic/intracellular and plasma-membrane-bound effector systems. The coupling efficiency and the plasma membrane localization of GPCRs are regulated by a variety of interacting proteins. In this review, we discuss recently disclosed protein interactions found with the cytoplasmic C-terminal tail regions of two types of presynaptic neurotransmitter receptors, the group III metabotropic glutamate receptors and the gamma-aminobutyric acid type-B receptors (GABA(B)Rs). Calmodulin binding to mGluR7 and other group III mGluRs may provide a Ca(2+)-dependent switch for unidirectional (G(alpha)) versus bidirectional (G(alpha) and G(beta)gamma) signalling to downstream effector proteins. In addition, clustering of mGluR7 by PICK1 (protein interacting with C-kinase 1), a polyspecific PDZ (PSD-95/Dlg1/ZO-1) domain containing synaptic organizer protein, sheds light on how higher-order receptor complexes with regulatory enzymes (or 'signalosomes') could be formed. The interaction of GABA(B)Rs with the adaptor protein 14-3-3 and the transcription factor ATF4 (activating transcription factor 4) suggests novel regulatory pathways for G-protein signalling, cytoskeletal reorganization and nuclear gene expression: processes that may all contribute to synaptic plasticity. PMID:12006104

  2. Myristoylated and non-myristoylated forms of the pH sensor protein hisactophilin II: intracellular shuttling to plasma membrane and nucleus monitored in real time by a fusion with green fluorescent protein.

    PubMed Central

    Hanakam, F; Albrecht, R; Eckerskorn, C; Matzner, M; Gerisch, G

    1996-01-01

    Hisactophilins are myristoylated proteins that are rich in histidine residues and known to exist in Dictyostelium cells in a plasma membrane-bound and a soluble cytoplasmic state. Intracellular translocation of these proteins in response to pH changes was monitored using hisactophilin fusions with green fluorescent protein (GFP) and confocal laser scanning microscopy. Both the normal and a mutated non-myristoylated fusion protein shuffled within the cells in a pH-dependent manner. After lowering the pH, these proteins translocated within minutes between the cytoplasm, the plasma membrane and the nucleus. The role of histidine clusters on the surface of hisactophilin molecules in binding of the proteins to the plasma membrane and in their transfer to the nucleus is discussed on the basis of a pH switch mechanism. Images PMID:8670794

  3. Identification of putative drug targets in Vancomycin-resistant Staphylococcus aureus (VRSA) using computer aided protein data analysis.

    PubMed

    Hasan, Md Anayet; Khan, Md Arif; Sharmin, Tahmina; Hasan Mazumder, Md Habibul; Chowdhury, Afrin Sultana

    2016-01-01

    Vancomycin-resistant Staphylococcus aureus (VRSA) is a Gram-positive, facultative aerobic bacterium which is evolved from the extensive exposure of Vancomycin to Methicillin resistant S. aureus (MRSA) that had become the most common cause of hospital and community-acquired infections. Due to the emergence of different antibiotic resistance strains, there is an exigency to develop novel drug targets to address the provocation of multidrug-resistant bacteria. In this study, in-silico genome subtraction methodology was used to design potential and pathogen specific drug targets against VRSA. Our study divulged 1987 proteins from the proteome of 34,549 proteins, which have no homologues in human genome after sequential analysis through CD-HIT and BLASTp. The high stringency analysis of the remaining proteins against database of essential genes (DEG) resulted in 169 proteins which are essential for S. aureus. Metabolic pathway analysis of human host and pathogen by KAAS at the KEGG server sorted out 19 proteins involved in unique metabolic pathways. 26 human non-homologous membrane-bound essential proteins including 4 which were also involved in unique metabolic pathway were deduced through PSORTb, CELLO v.2.5, ngLOC. Functional classification of uncharacterized proteins through SVMprot derived 7 human non-homologous membrane-bound hypothetical essential proteins. Study of potential drug target against Drug Bank revealed pbpA-penicillin-binding protein 1 and hypothetical protein MQW_01796 as the best drug target candidate. 2D structure was predicted by PRED-TMBB, 3D structure and functional analysis was also performed. Protein-protein interaction network of potential drug target proteins was analyzed by using STRING. The identified drug targets are expected to have great potential for designing novel drugs against VRSA infections and further screening of the compounds against these new targets may result in the discovery of novel therapeutic compounds that can be

  4. Engineering and overexpression of periplasmic forms of the penicillin-binding protein 3 of Escherichia coli.

    PubMed Central

    Fraipont, C; Adam, M; Nguyen-Distèche, M; Keck, W; Van Beeumen, J; Ayala, J A; Granier, B; Hara, H; Ghuysen, J M

    1994-01-01

    Replacement of the 36 and 56 N-terminal amino acid residues of the 588-amino-acid-residue membrane-bound penicillin-binding protein 3 (PBP3) of Escherichia coli by the OmpA signal peptide allows export of F37-V577 PBP3 and G57-V577 PBP3 respectively into the periplasm. The modified ftsI genes were placed under the control of the fused lpp promoter and lac promoter/operator; expression of the truncated PBP3s was optimized by varying the copy number of the recombinant plasmids and the amount of LacI repressor, and export was facilitated by increasing the SecB content of the producing strain. The periplasmic PBP3s (yield 8 mg/l of culture) were purified to 70% protein homogeneity. They require the presence of 0.25 M NaCl to remain soluble. Like the membrane-bound PBP3, they undergo processing by elimination of the C-terminal decapeptide I578-S588, they bind penicillin in a 1:1 molar ratio and they catalyse hydrolysis and aminolysis of acyclic thioesters that are analogues of penicillin. The membrane-anchor-free PBP3s have ragged N-termini. The G57-V577 PBP3, however, is less prone to proteolytic degradation than the F37-V577 PBP3. Images Figure 3 PMID:8129719

  5. Synthesis and localization of a development-specific protein in sclerotia of Sclerotinia sclerotiorum.

    PubMed Central

    Russo, G M; Van Etten, J L

    1985-01-01

    A development-specific protein (SSP) makes up about 35 to 40% of the total protein in sclerotia of the fungus Sclerotinia sclerotiorum. The protein consists of three charge isomers, with one isomer making up 80 to 90% of the total. In vitro translation of poly(A)+ RNA isolated from cells in early stages of sclerotia formation revealed that 44% of the amino acids incorporated was into SSP. In vivo- and in vitro-synthesized forms of SSP migrated at identical rates on both isoelectric focusing and denaturing polyacrylamide gels, indicating that SSP was not synthesized as a larger precursor. This was significant because SSP accumulated in membrane-bound, organellelike structures which resemble protein bodies found in seeds of many higher plants. Images PMID:2410402

  6. Communication: Microsecond dynamics of the protein and water affect electron transfer in a bacterial bc{sub 1} complex

    SciTech Connect

    Martin, Daniel R.; Matyushov, Dmitry V.

    2015-04-28

    Cross-membrane electron transport between cofactors localized in proteins of mitochondrial respiration and bacterial photosynthesis is the source of all biological energy. The statistics and dynamics of nuclear fluctuations in these protein/membrane/water heterogeneous systems are critical for their energetic efficiency. The results of 13 μs of atomistic molecular dynamics simulations of the membrane-bound bc{sub 1} bacterial complex are analyzed here. The reaction is affected by a broad spectrum of nuclear modes, with the slowest dynamics in the range of time-scales ∼0.1-1.6 μs contributing half of the reaction reorganization energy. Two reorganization energies are required to describe protein electron transfer due to dynamical arrest of protein conformations on the observation window. This mechanistic distinction allows significant lowering of activation barriers for reactions in proteins.

  7. A historical perspective on the lateral diffusion model of GTPase activation and related coupling of membrane signaling proteins

    PubMed Central

    Liebman, Paul A

    2014-01-01

    Aspects of our discovery of lateral diffusion of the G protein coupled receptor (GPCR) rhodopsin and that a single activated rhodopsin can non-covalently catalyze GTP binding to thousands of GTPases per second on rod disk membranes via this diffusion are summarized herein. Rapid GTPase coupling to membrane-bound phosphodiesterase (PDE) further amplifies the signal via cGMP hydrolysis, essential to visual transduction. Important generalizations from this work are that biomembranes can uniquely concentrate, orient for reaction and provide a solvent appropriate to rapid, powerful and appropriately controlled sequential interaction of signaling proteins. Of equal importance to function is timely control and termination of such powerful amplification via receptor phosphorylation (quenching) and arrestin binding. Downstream kinetic modulation by GTPase activating proteins (GAPs) and regulators of G protein signaling (RGS) and related mechanisms as well as limitations set by membrane domain fencing, structural protein binding etc. can be essential in relevant systems. PMID:25279248

  8. Hansenula polymorpha Pex1p and Pex6p are peroxisome-associated AAA proteins that functionally and physically interact.

    PubMed

    Kiel, J A; Hilbrands, R E; van der Klei, I J; Rasmussen, S W; Salomons, F A; van der Heide, M; Faber, K N; Cregg, J M; Veenhuis, M

    1999-08-01

    We have cloned the Hansenula polymorpha PEX1 and PEX6 genes by functional complementation of the corresponding peroxisome-deficient (pex) mutants. The gene products, HpPex1p and HpPex6p, are ATPases which both belong to the AAA protein family. Cells deleted for either gene (Deltapex1 or Deltapex6) were characterized by the presence of small peroxisomal remnants which contained peroxisomal membrane proteins and minor amounts of matrix proteins. The bulk of the matrix proteins, however, resided in the cytosol. In cell fractionation studies HpPex1p and HpPex6p co-sedimented with the peroxisomal membrane protein HpPex3p in both wild-type cells and in Deltapex4, Deltapex8 or Deltapex14 cells. Both proteins are loosely membrane-bound and face the cytosol. Furthermore, HpPex1p and HpPex6p physically and functionally interact in vivo. Overexpression of PEX6 resulted in defects in peroxisomal matrix protein import. By contrast, overexpression of PEX1 was not detrimental to the cells. Interestingly, co-overproduction of HpPex1p rescued the protein import defect caused by HpPex6p overproduction. Overproduced HpPex1p and HpPex6p remained predominantly membrane-bound, but only partially co-localized with the peroxisomal membrane protein HpPex3p. Our data indicate that HpPex1p and HpPex6p function in a protein complex associated with the peroxisomal membrane and that overproduced, mislocalized HpPex6p prevents HpPex1p from reaching its site of activity. PMID:10455230

  9. Altered Expression of Bone Morphogenetic Protein Accessory Proteins in Murine and Human Pulmonary Fibrosis.

    PubMed

    Murphy, Noelle; Gaynor, Katherine U; Rowan, Simon C; Walsh, Sinead M; Fabre, Aurelie; Boylan, John; Keane, Michael P; McLoughlin, Paul

    2016-03-01

    Idiopathic pulmonary fibrosis is a chronic, progressive fibrotic disease with a poor prognosis. The balance between transforming growth factor β1 and bone morphogenetic protein (BMP) signaling plays an important role in tissue homeostasis, and alterations can result in pulmonary fibrosis. We hypothesized that multiple BMP accessory proteins may be responsible for maintaining this balance in the lung. Using the bleomycin mouse model for fibrosis, we examined an array of BMP accessory proteins for changes in mRNA expression. We report significant increases in mRNA expression of gremlin 1, noggin, follistatin, and follistatin-like 1 (Fstl1), and significant decreases in mRNA expression of chordin, kielin/chordin-like protein, nephroblastoma overexpressed gene, and BMP and activin membrane-bound inhibitor (BAMBI). Protein expression studies demonstrated increased levels of noggin, BAMBI, and FSTL1 in the lungs of bleomycin-treated mice and in the lungs of idiopathic pulmonary fibrosis patients. Furthermore, we demonstrated that transforming growth factor β stimulation resulted in increased expression of noggin, BAMBI, and FSTL1 in human small airway epithelial cells. These results provide the first evidence that multiple BMP accessory proteins are altered in fibrosis and may play a role in promoting fibrotic injury. PMID:26765958

  10. Identification of Protein-Protein Interactions and Topologies in Living Cells with Chemical Cross-linking and Mass Spectrometry*S⃞

    PubMed Central

    Zhang, Haizhen; Tang, Xiaoting; Munske, Gerhard R.; Tolic, Nikola; Anderson, Gordon A.; Bruce, James E.

    2009-01-01

    We present results from a novel strategy that enables concurrent identification of protein-protein interactions and topologies in living cells without specific antibodies or genetic manipulations for immuno-/affinity purifications. The strategy consists of (i) a chemical cross-linking reaction: intact cell labeling with a novel class of chemical cross-linkers, protein interaction reporters (PIRs); (ii) two-stage mass spectrometric analysis: stage 1 identification of PIR-labeled proteins and construction of a restricted database by two-dimensional LC/MSMS and stage 2 analysis of PIR-labeled peptides by multiplexed LC/FTICR-MS; and (iii) data analysis: identification of cross-linked peptides and proteins of origin using accurate mass and other constraints. The primary advantage of the PIR approach and distinction from current technology is that protein interactions together with topologies are detected in native biological systems by stabilizing protein complexes with new covalent bonds while the proteins are present in the original cellular environment. Thus, weak or transient interactions or interactions that require properly folded, localized, or membrane-bound proteins can be labeled and identified through the PIR approach. This strategy was applied to Shewanella oneidensis bacterial cells, and initial studies resulted in identification of a set of protein-protein interactions and their contact/binding regions. Furthermore most identified interactions involved membrane proteins, suggesting that the PIR approach is particularly suited for studies of membrane protein-protein interactions, an area under-represented with current widely used approaches. PMID:18936057

  11. The membrane attack complex, perforin and cholesterol-dependent cytolysin superfamily of pore-forming proteins.

    PubMed

    Lukoyanova, Natalya; Hoogenboom, Bart W; Saibil, Helen R

    2016-06-01

    The membrane attack complex and perforin proteins (MACPFs) and bacterial cholesterol-dependent cytolysins (CDCs) are two branches of a large and diverse superfamily of pore-forming proteins that function in immunity and pathogenesis. During pore formation, soluble monomers assemble into large transmembrane pores through conformational transitions that involve extrusion and refolding of two α-helical regions into transmembrane β-hairpins. These transitions entail a dramatic refolding of the protein structure, and the resulting assemblies create large holes in cellular membranes, but they do not use any external source of energy. Structures of the membrane-bound assemblies are required to mechanistically understand and modulate these processes. In this Commentary, we discuss recent advances in the understanding of assembly mechanisms and molecular details of the conformational changes that occur during MACPF and CDC pore formation. PMID:27179071

  12. Protein transport across the parasitophorous vacuole of Plasmodium falciparum: into the great wide open.

    PubMed

    Charpian, Stefan; Przyborski, Jude M

    2008-02-01

    The human malaria parasite Plasmodium falciparum resides and multiplies within a membrane-bound vacuole in the cytosol of its host cell, the mature human erythrocyte. To enable the parasite to complete its intraerythrocytic life cycle, a large number of parasite proteins are synthesized and transported from the parasite to the infected cell. To gain access to the erythrocyte, parasite proteins must first cross the membrane of the parasitophorous vacuole (PVM), a process that is not well understood at the mechanistic level. Here, we review past and current literature on this topic, and make tentative predictions about the nature of the transport machinery required for transport of proteins across the PVM, and the molecular factors involved. PMID:17944805

  13. Size-dependent protein segregation at membrane interfaces

    NASA Astrophysics Data System (ADS)

    Schmid, Eva M.; Bakalar, Matthew H.; Choudhuri, Kaushik; Weichsel, Julian; Ann, Hyoung Sook; Geissler, Phillip L.; Dustin, Michael L.; Fletcher, Daniel A.

    2016-07-01

    Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane proteins whose organization is critical for intracellular signalling. To isolate the role of membrane protein size in pattern formation, we reconstituted model membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between membrane proteins can drastically alter their organization at membrane interfaces, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally driven membrane height fluctuations that transiently limit access to the interface. This sensitive and highly effective means of physically segregating proteins has implications for cell-cell contacts such as T-cell immunological synapses (for example, CD45 exclusion) and epithelial cell junctions (for example, E-cadherin enrichment), as well as for protein sorting at intracellular contact points between membrane-bound organelles.

  14. Epitope mapping of 10 monoclonal antibodies against the pig analogue of human membrane cofactor protein (MCP)

    PubMed Central

    PéRez De La Lastra, J M; Van Den Berg, C W; Bullido, R; Almazán, F; Domínguez, J; Llanes, D; Morgan, B P

    1999-01-01

    Pig membrane cofactor protein (MCP; CD46) is a 50 000–60 000 MW glycoprotein that is expressed on a wide variety of cells, including erythrocytes. Pig MCP has cofactor activity for factor I-mediated cleavage of C3b and is an efficient regulator of the classical and alternative pathway of human and pig complement. A panel of 10 monoclonal antibodies (mAbs) was collected from two different laboratories; all of these mAbs were raised against pig leucocytes and all recognized the same complex banding pattern on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) of erythrocyte membranes. All were shown to be reactive with pig MCP and were divided into four groups of mutually competitive antibodies based on competition studies for membrane-bound MCP and for soluble MCP, the latter by surface plasmon resonance (SPR) analysis. The antigenic properties of membrane-bound and soluble MCP were similar, although some interesting differences were revealed. None of the 10 mAbs were cross-reactive with human MCP and only one showed cross-reactivity with leucocytes from a panel of large mammals – a weak cross-reactivity with a subset of dog leucocytes. All antibodies in one of the epitope groups and some in a second epitope group were able to block the functional activity of pig MCP, as measured by inhibition of MCP-catalysed C3 degradation by factor I. PMID:10233756

  15. Charged gels as orienting media for measurement of residual dipolar couplings in soluble and integral membrane proteins.

    PubMed

    Cierpicki, Tomasz; Bushweller, John H

    2004-12-15

    Measurement of residual dipolar couplings for membrane proteins will dramatically improve the quality of the structures obtainable by solution NMR spectroscopy. While there has been some success in achieving alignment of membrane-bound peptides, there has been very limited success in achieving alignment for functional membrane proteins. Herein, we demonstrate that charged polyacrylamide-based copolymers are suitable for obtaining weak alignment of membrane proteins reconstituted in detergent micelles. Varying the copolymer compositions, we prepared positively, zwitterionic, and negatively charged gels that are very stable at low concentration and can be used for obtaining weak alignment by compression in an NMR tube. Application of this method is demonstrated for the integral membrane protein OmpA in DPC micelles. PMID:15584763

  16. Effect of IL-6 and IL-8 on the expression of the complement activation inhibitors MAC-inhibitory protein and decay-accelerating factor in ovarian cancer A2780 cells

    PubMed Central

    Kapka-Skrzypczak, Lucyna; Popek, Sylwia; Sawicki, Krzysztof; Wolińska, Ewa; Czajka, Magdalena; Skrzypczak, Maciej

    2016-01-01

    The aim of the present study was to evaluate the role of interleukin (IL)-6 and IL-8 on the expression of the membrane-bound complement inhibitors membrane attack complex-inhibitory protein (CD59) and decay-accelerating factor (CD55), in the human ovarian carcinoma A2780 cell line, which is a non-producing IL-6 cell line that does exhibit IL-6 responsiveness, due to the presence of IL-6 receptors. Extracellular levels of complement system inhibitors were evaluated by western blotting and reverse transcription-quantitative polymerase chain reaction. Cellular localization of CD55 and CD59 in the ovarian cancer cells was assessed by immunofluorescence. The detection of a soluble form of CD55 and CD59 released by the A2780 cells following stimulation with IL-6 and IL-8 was detected by enzyme-linked immunosorbent assay. The present data revealed that A2780 cells express CD55 and CD59 at the mRNA and protein level, but do not secrete these proteins to the culture medium. Results of western blotting demonstrated that the protein level of CD59 was regulated by IL-6 and IL-8 in a dose-dependent manner. Immunofluorescence analysis revealed that the ovarian cancer A2780 cell line expresses the membrane bound form of CD55 protein. The present results indicate that CD55 and CD59 may affect the efficiency of complement-mediated immunotherapies. PMID:27446461

  17. Assessment of biological characteristics of adipose tissue-derived stem cells co-labeled with Molday ION Rhodamine B™ and green fluorescent protein in vitro.

    PubMed

    Nan, Hua; Huang, Jiacheng; Li, Hongmian; Li, Qiong; Liu, Dalie

    2013-11-01

    The current study aimed to investigate adipose tissue-derived stem cells (ADSCs) in vivo by multimodality imaging following implantation for cellular therapy. The biological characteristics of ADSCs co-labeled with Molday ION Rhodamine B™ (MIRB) and green fluorescent protein (GFP) were studied in vitro. Following rat ADSC isolation and culture, a combined labeling strategy for ADSCs based on genetic modification of the reporter gene GFP with lentiviral vector expression enhancement and physical MIRB labeling was performed. Cell viability, proliferation, membrane-bound antigens and multiple differentiation ability were compared between the labeled and unlabeled ADSCs. The ADSCs were successfully labeled with GFP and MIRB, showing various fluorescent colors for marker identification. The fluorescence emitted by the GFP protein was sustained and exhibited stable expression, while MIRB fluorescence decreased with time. Compared with the unlabeled ADSCs, no significant differences were detected in cell viability, proliferation, membrane-bound antigens and multiple differentiation ability in the co-labeled samples (P>0.05). No significant effects on the biophysical properties of ADSCs were observed following co-labeling with lentiviral vectors encoding the gene for emerald green fluorescent protein and MIRB. The ADSCs were able to be efficiently tracked in vitro and in vivo by multimodality imaging thus, the co-labeling approach provides a novel strategy for therapeutic gene studies. PMID:24065138

  18. Registration of the protein with compact disk.

    PubMed

    Ivanov, Yuri Dmitrievich; Pleshakova, Tatyana Olegovna; Krohin, Nikolay Valentinovich; Kaysheva, Anna Leonidovna; Usanov, Sergey Alexandrovich; Archakov, Alexander Ivanovich

    2013-05-15

    CD-based optico-acoustical biosensor (OAB) was used for detection of various types of proteins represented by bovine serum albumin (BSA), heme-containing myoglobin (Mb), monoclonal antibody against viral protein marker of hepatitis B (anti-HBsAg) and membrane-bound cytochrome P450scc (P450scc). We applied standard compact disc reader (CD-ROM) as an optical analyzer and a standard compact disc (CD) as a biochip containing immobilized protein molecules. This biosensor can translate into a digital code the changes of optical signal from the proteins and their complexes immobilized on the CD surface. Then, the digital code is translated into an acoustic series or, in other words, into a "music of proteins". We demonstrate the use of the OAB for direct detection of proteins with different molecular weights, such as BSA, Mb, P450scc, anti-HBsAg with the concentration detection limit (DL) about 10(-7)M. By signal amplification achieved with autometallography, a higher sensitivity level (DL∼10(-9)M) for the detection of myoglobin was obtained. The method of OAB-detection of proteins is cheap: it requires no special equipment like spectrometers, refractometers and other devices. Due to the fact that acoustic series of the protein complexes antigen/antibody differs from that of single proteins, the OAB-detection is of particular interest for rapid assay in yes/no data type and for home diagnostics. Combination of the OAB with a mass spectrometer allowed the detection and identification of the target proteins fished out directly onto a standard CD surface. PMID:23357004

  19. Topological Analysis of Hedgehog Acyltransferase, a Multipalmitoylated Transmembrane Protein*

    PubMed Central

    Konitsiotis, Antonio D.; Jovanović, Biljana; Ciepla, Paulina; Spitaler, Martin; Lanyon-Hogg, Thomas; Tate, Edward W.; Magee, Anthony I.

    2015-01-01

    Hedgehog proteins are secreted morphogens that play critical roles in development and disease. During maturation of the proteins through the secretory pathway, they are modified by the addition of N-terminal palmitic acid and C-terminal cholesterol moieties, both of which are critical for their correct function and localization. Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmic reticulum that palmitoylates Hedgehog proteins, is a member of a small subfamily of membrane-bound O-acyltransferase proteins that acylate secreted proteins, and is an important drug target in cancer. However, little is known about HHAT structure and mode of function. We show that HHAT is comprised of ten transmembrane domains and two reentrant loops with the critical His and Asp residues on opposite sides of the endoplasmic reticulum membrane. We further show that HHAT is palmitoylated on multiple cytosolic cysteines that maintain protein structure within the membrane. Finally, we provide evidence that mutation of the conserved His residue in the hypothesized catalytic domain results in a complete loss of HHAT palmitoylation, providing novel insights into how the protein may function in vivo. PMID:25505265

  20. Topological analysis of Hedgehog acyltransferase, a multipalmitoylated transmembrane protein.

    PubMed

    Konitsiotis, Antonio D; Jovanović, Biljana; Ciepla, Paulina; Spitaler, Martin; Lanyon-Hogg, Thomas; Tate, Edward W; Magee, Anthony I

    2015-02-01

    Hedgehog proteins are secreted morphogens that play critical roles in development and disease. During maturation of the proteins through the secretory pathway, they are modified by the addition of N-terminal palmitic acid and C-terminal cholesterol moieties, both of which are critical for their correct function and localization. Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmic reticulum that palmitoylates Hedgehog proteins, is a member of a small subfamily of membrane-bound O-acyltransferase proteins that acylate secreted proteins, and is an important drug target in cancer. However, little is known about HHAT structure and mode of function. We show that HHAT is comprised of ten transmembrane domains and two reentrant loops with the critical His and Asp residues on opposite sides of the endoplasmic reticulum membrane. We further show that HHAT is palmitoylated on multiple cytosolic cysteines that maintain protein structure within the membrane. Finally, we provide evidence that mutation of the conserved His residue in the hypothesized catalytic domain results in a complete loss of HHAT palmitoylation, providing novel insights into how the protein may function in vivo. PMID:25505265

  1. Analysis of interactions between SNARE proteins using imaging ellipsometer coupled with microfluidic array

    PubMed Central

    Qi, Cai; Zhang, Hong; Liu, Li; Yang, Yinke; Kang, Tengfei; Hao, Wenxin; Jin, Gang; Jiang, Taijiao

    2014-01-01

    The soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins are small and abundant membrane-bound proteins, whose specific interactions mediate membrane fusion during cell fusion or cellular trafficking. In this study, we report the use of a label-free method, called imaging ellipsometer to analyze the interactions among three SNAREs, namely Sec22p, Ykt6p and Sso2p. The SNAREs were immobilized on the silicon wafer and then analyzed in a pairwise mode with microfluidic array, leading us to discover the interactions between Ykt6p and Sso2p, Sec22p and Sso2p. Moreover, by using the real-time function of the imaging ellipsometer, we were able to obtain their association constants (KA) of about 104 M−1. We argue that the use of imaging ellipsometer coupled with microfluidic device will deepen our understanding of the molecular mechanisms underlying membrane fusion process. PMID:24938428

  2. Mass-spectrometry data for Rhizoctonia solani proteins produced during infection of wheat and vegetative growth.

    PubMed

    Anderson, Jonathan P; Hane, James K; Stoll, Thomas; Pain, Nicholas; Hastie, Marcus L; Kaur, Parwinder; Hoogland, Christine; Gorman, Jeffrey J; Singh, Karam B

    2016-09-01

    Rhizoctonia solani is an important root infecting pathogen of a range of food staples worldwide including wheat, rice, maize, soybean, potato, legumes and others. Conventional resistance breeding strategies are hindered by the absence of tractable genetic resistance in any crop host. Understanding the biology and pathogenicity mechanisms of this fungus is important for addressing these disease issues, however, little is known about how R. solani causes disease. The data described in this article is derived from applying mass spectrometry based proteomics to identify soluble, membrane-bound and culture filtrate proteins produced under wheat infection and vegetative growth conditions. Comparisons of the data for sample types in this set will be useful to identify metabolic pathway changes as the fungus switches from saprophytic to a pathogenic lifestyle or pathogenicity related proteins contributing to the ability to cause disease on wheat. The data set is deposited in the PRIDE archive under identifier PRIDE: PXD002806. PMID:27331100

  3. Membrane protein biosensing with plasmonic nanopore arrays and pore-spanning lipid membranes

    PubMed Central

    Im, Hyungsoon; Wittenberg, Nathan J.; Lesuffleur, Antoine; Lindquist, Nathan C.; Oh, Sang-Hyun

    2010-01-01

    Integration of solid-state biosensors and lipid bilayer membranes is important for membrane protein research and drug discovery. In these sensors, it is critical that the solid-state sensing material does not have adverse effects on the conformation or functionality of membrane-bound molecules. In this work, pore-spanning lipid membranes are formed over an array of periodic nanopores in free-standing gold films for surface plasmon resonance (SPR) kinetic binding assays. The ability to perform kinetic assays with a transmembrane protein is demonstrated with α-hemolysin (α-HL). The incorporation of α-HL into the membrane followed by specific antibody binding (anti-α-HL) red-shifts the plasmon resonance of the gold nanopore array, which is optically monitored in real time. Subsequent fluorescence imaging reveals that the antibodies primarily bind in nanopore regions, indicating that α-HL incorporation preferentially occurs into areas of pore-spanning lipid membranes. PMID:21218136

  4. Lipid-mediated Protein-protein Interactions Modulate Respiration-driven ATP Synthesis

    PubMed Central

    Nilsson, Tobias; Lundin, Camilla Rydström; Nordlund, Gustav; Ädelroth, Pia; von Ballmoos, Christoph; Brzezinski, Peter

    2016-01-01

    Energy conversion in biological systems is underpinned by membrane-bound proton transporters that generate and maintain a proton electrochemical gradient across the membrane which used, e.g. for generation of ATP by the ATP synthase. Here, we have co-reconstituted the proton pump cytochrome bo3 (ubiquinol oxidase) together with ATP synthase in liposomes and studied the effect of changing the lipid composition on the ATP synthesis activity driven by proton pumping. We found that for 100 nm liposomes, containing 5 of each proteins, the ATP synthesis rates decreased significantly with increasing fractions of DOPA, DOPE, DOPG or cardiolipin added to liposomes made of DOPC; with e.g. 5% DOPG, we observed an almost 50% decrease in the ATP synthesis rate. However, upon increasing the average distance between the proton pumps and ATP synthases, the ATP synthesis rate dropped and the lipid dependence of this activity vanished. The data indicate that protons are transferred along the membrane, between cytochrome bo3 and the ATP synthase, but only at sufficiently high protein densities. We also argue that the local protein density may be modulated by lipid-dependent changes in interactions between the two proteins complexes, which points to a mechanism by which the cell may regulate the overall activity of the respiratory chain. PMID:27063297

  5. Kinetic Measurements Reveal Enhanced Protein-Protein Interactions at Intercellular Junctions

    PubMed Central

    Shashikanth, Nitesh; Kisting, Meridith A.; Leckband, Deborah E.

    2016-01-01

    The binding properties of adhesion proteins are typically quantified from measurements with soluble fragments, under conditions that differ radically from the confined microenvironment of membrane bound proteins in adhesion zones. Using classical cadherin as a model adhesion protein, we tested the postulate that confinement within quasi two-dimensional intercellular gaps exposes weak protein interactions that are not detected in solution binding assays. Micropipette-based measurements of cadherin-mediated, cell-cell binding kinetics identified a unique kinetic signature that reflects both adhesive (trans) bonds between cadherins on opposing cells and lateral (cis) interactions between cadherins on the same cell. In solution, proposed lateral interactions were not detected, even at high cadherin concentrations. Mutations postulated to disrupt lateral cadherin association altered the kinetic signatures, but did not affect the adhesive (trans) binding affinity. Perturbed kinetics further coincided with altered cadherin distributions at junctions, wound healing dynamics, and paracellular permeability. Intercellular binding kinetics thus revealed cadherin interactions that occur within confined, intermembrane gaps but not in solution. Findings further demonstrate the impact of these revealed interactions on the organization and function of intercellular junctions. PMID:27009566

  6. Kinetic Measurements Reveal Enhanced Protein-Protein Interactions at Intercellular Junctions.

    PubMed

    Shashikanth, Nitesh; Kisting, Meridith A; Leckband, Deborah E

    2016-01-01

    The binding properties of adhesion proteins are typically quantified from measurements with soluble fragments, under conditions that differ radically from the confined microenvironment of membrane bound proteins in adhesion zones. Using classical cadherin as a model adhesion protein, we tested the postulate that confinement within quasi two-dimensional intercellular gaps exposes weak protein interactions that are not detected in solution binding assays. Micropipette-based measurements of cadherin-mediated, cell-cell binding kinetics identified a unique kinetic signature that reflects both adhesive (trans) bonds between cadherins on opposing cells and lateral (cis) interactions between cadherins on the same cell. In solution, proposed lateral interactions were not detected, even at high cadherin concentrations. Mutations postulated to disrupt lateral cadherin association altered the kinetic signatures, but did not affect the adhesive (trans) binding affinity. Perturbed kinetics further coincided with altered cadherin distributions at junctions, wound healing dynamics, and paracellular permeability. Intercellular binding kinetics thus revealed cadherin interactions that occur within confined, intermembrane gaps but not in solution. Findings further demonstrate the impact of these revealed interactions on the organization and function of intercellular junctions. PMID:27009566

  7. CHAPS solubilization of a G-protein sensitive 5-HT sub 1 A receptor from bovine hippocampus

    SciTech Connect

    Kline, T. ); Park, H.; Meyerson, L.R. )

    1989-01-01

    The binding of ({sup 3}H) 8-OH-DPAT to membrane-bound 5-HT{sub 1A} receptors from bovine hippocampus was saturable and corresponded to a single high-affinity state. Solubilization of the bovine hippocampal membranes with 10 mM CHAPS containing 200 mM NaCl, renders a preparation which binds ({sup 3}H) 8-OH-DPAT with high affinity and is guanine nucleotide sensitive and ketanserin insensitive. 50% of ({sup 3}H) 8-OH-DPAT binding activity is solubilized. The presence of GMP-P(NH)P promotes a low-affinity state which is characteristic of receptors coupled to G-proteins. GMP-P(NH)P markedly accelerates the dissociation ({sup 3}H) 8-OH-DPAT from solubilized membranes while having negligible effects on association. Thus, the agonist can activate the ternary complex rather than to promote its formation. 8-OH-DPAT, WB 4101 and 5-carboxamidotryptamine dose responsively inhibit soluble ({sup 3}H) 8-OH-DPAT binding with IC{sub 50} values of 16.1, 15.6 and 1.3 nM, respectively. The CHAPS solubilized membrane preparation retains many of the ({sup 3}H) 8-OH-DPAT binding characteristics of the membrane bound form.

  8. Geometry-induced protein pattern formation

    PubMed Central

    Thalmeier, Dominik; Halatek, Jacob; Frey, Erwin

    2016-01-01

    Protein patterns are known to adapt to cell shape and serve as spatial templates that choreograph downstream processes like cell polarity or cell division. However, how can pattern-forming proteins sense and respond to the geometry of a cell, and what mechanistic principles underlie pattern formation? Current models invoke mechanisms based on dynamic instabilities arising from nonlinear interactions between proteins but neglect the influence of the spatial geometry itself. Here, we show that patterns can emerge as a direct result of adaptation to cell geometry, in the absence of dynamical instability. We present a generic reaction module that allows protein densities robustly to adapt to the symmetry of the spatial geometry. The key component is an NTPase protein that cycles between nucleotide-dependent membrane-bound and cytosolic states. For elongated cells, we find that the protein dynamics generically leads to a bipolar pattern, which vanishes as the geometry becomes spherically symmetrical. We show that such a reaction module facilitates universal adaptation to cell geometry by sensing the local ratio of membrane area to cytosolic volume. This sensing mechanism is controlled by the membrane affinities of the different states. We apply the theory to explain AtMinD bipolar patterns in Δ EcMinDE Escherichia coli. Due to its generic nature, the mechanism could also serve as a hitherto-unrecognized spatial template in many other bacterial systems. Moreover, the robustness of the mechanism enables self-organized optimization of protein patterns by evolutionary processes. Finally, the proposed module can be used to establish geometry-sensitive protein gradients in synthetic biological systems. PMID:26739566

  9. Distinct roles of activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) in transcription during the mammalian unfolded protein response.

    PubMed Central

    Okada, Tetsuya; Yoshida, Hiderou; Akazawa, Rieko; Negishi, Manabu; Mori, Kazutoshi

    2002-01-01

    In response to accumulation of unfolded proteins in the endoplasmic reticulum (ER), a homoeostatic response, termed the unfolded protein response (UPR), is activated in all eukaryotic cells. The UPR involves only transcriptional regulation in yeast, and approx. 6% of all yeast genes, encoding not only proteins to augment the folding capacity in the ER, but also proteins working at various stages of secretion, are induced by ER stress [Travers, Patil, Wodicka, Lockhart, Weissman and Walter (2000) Cell (Cambridge, Mass.) 101, 249-258]. In the present study, we conducted microarray analysis of HeLa cells, although our analysis covered only a small fraction of the human genome. A great majority of human ER stress-inducible genes (approx. 1% of 1800 genes examined) were classified into two groups. One group consisted of genes encoding ER-resident molecular chaperones and folding enzymes, and these genes were directly regulated by the ER-membrane-bound transcription factor activating transcription factor (ATF) 6. The ER-membrane-bound protein kinase double-stranded RNA-activated protein kinase-like ER kinase (PERK)-mediated signalling pathway appeared to be responsible for induction of the remaining genes, which are not involved in secretion, but may be important after cellular recovery from ER stress. In higher eukaryotes, the PERK-mediated translational-attenuation system is known to operate in concert with the transcriptional-induction system. Thus we propose that mammalian cells have evolved a strategy to cope with ER stress different from that of yeast cells. PMID:12014989

  10. Development of electron spin echo envelope modulation spectroscopy to probe the secondary structure of recombinant membrane proteins in a lipid bilayer.

    PubMed

    Zhang, Rongfu; Sahu, Indra D; Gibson, Kaylee R; Muhammad, Nefertiti B; Bali, Avnika P; Comer, Raven G; Liu, Lishan; Craig, Andrew F; Mccarrick, Robert M; Dabney-Smith, Carole; Sanders, Charles R; Lorigan, Gary A

    2015-11-01

    Membrane proteins conduct many important biological functions essential to the survival of organisms. However, due to their inherent hydrophobic nature, it is very difficult to obtain structural information on membrane-bound proteins using traditional biophysical techniques. We are developing a new approach to probe the secondary structure of membrane proteins using the pulsed EPR technique of Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy. This method has been successfully applied to model peptides made synthetically. However, in order for this ESEEM technique to be widely applicable to larger membrane protein systems with no size limitations, protein samples with deuterated residues need to be prepared via protein expression methods. For the first time, this study shows that the ESEEM approach can be used to probe the local secondary structure of a (2) H-labeled d8 -Val overexpressed membrane protein in a membrane mimetic environment. The membrane-bound human KCNE1 protein was used with a known solution NMR structure to demonstrate the applicability of this methodology. Three different α-helical regions of KCNE1 were probed: the extracellular domain (Val21), transmembrane domain (Val50), and cytoplasmic domain (Val95). These results indicated α-helical structures in all three segments, consistent with the micelle structure of KCNE1. Furthermore, KCNE1 was incorporated into a lipid bilayer and the secondary structure of the transmembrane domain (Val50) was shown to be α-helical in a more native-like environment. This study extends the application of this ESEEM approach to much larger membrane protein systems that are difficult to study with X-ray crystallography and/or NMR spectroscopy. PMID:26355804

  11. Role of lipids in the translocation of proteins across membranes.

    PubMed Central

    Van Voorst, F; De Kruijff, B

    2000-01-01

    The architecture of cells, with various membrane-bound compartments and with the protein synthesizing machinery confined to one location, dictates that many proteins have to be transported through one or more membranes during their biogenesis. A lot of progress has been made on the identification of protein translocation machineries and their sorting signals in various organelles and organisms. Biochemical characterization has revealed the functions of several individual protein components. Interestingly, lipid components were also found to be essential for the correct functioning of these translocases. This led to the idea that there is a very intimate relationship between the lipid and protein components that enables them to fulfil their intriguing task of transporting large biopolymers through a lipid bilayer without leaking their contents. In this review we focus on the Sec translocases in the endoplasmic reticulum and the bacterial inner membrane. We also highlight the interactions of lipids and proteins during the process of translocation and integrate this into a model that enables us to understand the role of membrane lipid composition in translocase function. PMID:10769162

  12. Protein crystals on phase-separating model membranes

    NASA Astrophysics Data System (ADS)

    Manley, Suliana; Horton, Margaret; Leszczynski, Szymon; Gast, Alice

    2006-03-01

    We study the interplay between the crystallization of proteins tethered to membranes and separation within the membranes of giant unilamellar vesicles (GUVs) composed of DOPC, sphingomyelin (SM), and cholesterol. These model membranes phase separate into coexisting liquid domains below a miscibility transition temperature. This phase separation captures some aspects of the formation of lipid rafts in cell membranes and demonstrates the influence of membrane composition on raft formation. Real cell membranes have a much more complicated structure. There are additional physical constraints present in cell membranes, such as their attachment to the cytoskeleton and the presence of membrane bound proteins. The self-association of membrane proteins can influence the membrane phase behavior. We begin to investigate these effects on model tethered protein- loaded membranes by incorporating a small amount of biotin-X- DPPE into our GUVs. The biotinylated lipid partitions into a cholesterol-poor phase; thus, streptavidin binds preferentially to one of the membrane phases. As streptavidin assembles to form crystalline domains, it restricts the membrane mobility. We examine the effect of this protein association on lipid phase separation, as well as the effect of the lipid phase separation on the crystallization of the tethered proteins.

  13. Antibody arrays for determination of relative protein abundances.

    PubMed

    Chaga, Grigoriy S

    2008-01-01

    As a large number of genome-sequencing projects reached completion, the attention of the scientific community is turning toward understanding the structure-functions of gene translation products-the proteins as well as the complete complement of proteins-the proteome. One goal of proteomics is to correlate changes in protein abundance with biological processes and disease states. To help accelerate this avenue of proteomics, a significant effort has been devoted to the development of multiplexed methods for protein analyses. We have developed an Antibody Microarray, a chip-based technology for multiparallel determination of relative abundance of hundreds of proteins. The Antibody Microarray is composed of hundreds of distinct monoclonal antibodies printed at high density on a glass slide. It utilizes a novel experimental setup and data analysis algorithm, which enables scientists to assay hundreds of cytosolic, nuclear, and membrane-bound proteins with a single experiment. Examples of biological samples that are analyzed on the Antibody Microarray include tissue samples, cell cultures, and body fluids. PMID:18370316

  14. Cell-Free Expression of G Protein-Coupled Receptors.

    PubMed

    Segers, Kenneth; Masure, Stefan

    2015-01-01

    The large-scale production of recombinant G protein-coupled receptors (GPCRs) is one of the major bottlenecks that hamper functional and structural studies of this important class of integral membrane proteins. Heterologous overexpression of GPCRs often results in low yields of active protein, usually due to a combination of several factors, such as low expression levels, protein insolubility, host cell toxicity, and the need to use harsh and often denaturing detergents (e.g., SDS, LDAO, OG, and DDM, among others) to extract the recombinant receptor from the host cell membrane. Many of these problematic issues are inherently linked to cell-based expression systems and can therefore be circumvented by the use of cell-free systems. In this unit, we provide a range of protocols for the production of GPCRs in a cell-free expression system. Using this system, we typically obtain GPCR expression levels of ∼1 mg per ml of reaction mixture in the continuous-exchange configuration. Although the protocols in this unit have been optimized for the cell-free expression of GPCRs, they should provide a good starting point for the production of other classes of membrane proteins, such as ion channels, aquaporins, carrier proteins, membrane-bound enzymes, and even large molecular complexes. PMID:26237676

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

    PubMed Central

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

    2015-01-01

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

  16. Site on the human erythrocyte glucose transporter phosphorylated by protein kinase C resides on the protein's hydrophilic domain

    SciTech Connect

    Deziel, M.R.; McReynolds, J.H.; Lippes, H.A.; Jung, C.Y.

    1986-05-01

    A recently published model of the human erythrocyte hexose transporter deduced from the protein's primary structure proposes that the transporter is organized into two membrane domains comprising 77% of the protein's mass and three hydrophilic domains, a short segment that includes the polypeptide's N-terminus and two larger segments, one lying between the membrane domains and the other at the protein's C-terminus. Limited tryptic digestion of the transporter produces two membrane-bound fragments corresponding to the proposed membrane domains and releases a number of soluble peptides. Fast Atom Bombardment Mass Spectroscopic analysis of the released peptides and comparison of the peptide's masses with the transporter's amino acid sequence revealed that tryptic peptides corresponding to at least 63% of the hydrophilic domains' mass were recovered. The site of phosphorylation by protein kinase C, tagged using (/sup 32/P)-ATP, was also released from the transporter under these conditions, (in contrast to sites located within the protein's membrane domains), indicating that this site is located within one of the hydrophilic domains. Tryptic digestion at elevated ionic strength or cleavage with S. Aureus V8 protease results in the recovery of the /sup 32/P label on the carbohydrate-bearing membrane domain that is located near the protein's N-terminus, thus eliminating the C-terminal hydrophilic segment as a possible site of phosphorylation.

  17. Agonist-induced ADP-ribosylation of a cytosolic protein in human platelets

    SciTech Connect

    Bruene, B.; Molina Y Vedia, L.; Lapetina, E.G. )

    1990-05-01

    {alpha}-Thrombin and phorbol 12,13-dibutyrate stimulated the mono(ADP-ribosyl)ation of a 42-kDa cytosolic protein of human platelets. This effect was mediated by protein kinase C activation and was inhibited by protein kinase C inhibitor staurosporine. It also was prevented by prostacyclin, which is known to inhibit the phospholipase C-induced formation of 1,2-diacylglycerol, which is one of the endogenous activators of protein kinase C. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the 42-kDa protein that is ADP-ribosylated by {alpha}-thrombin was clearly distinct from the {alpha} subunits of membrane-bound inhibitory and stimulatory guanine nucleotide-binding regulatory proteins, respectively G{sub i{alpha}} and G{sub s{alpha}}; the 47-kDa protein that is phosphorylated by protein kinase C in platelets; and the 39-kDa protein that has been shown to be endogenously ADP-ribosylated by agents that release nitric oxide. This information shows that agonist-induced activation of protein kinase leads to the ADP-ribosylation of a specific protein. This covalent modification might have a functional role in platelet activation.

  18. Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins

    PubMed Central

    Klein, Cornelia; Terrao, Monica; Inchaustegui Gil, Diana; Clayton, Christine

    2015-01-01

    We report here the results of experiments designed to identify RNA-binding proteins that might be associated with Trypanosoma brucei polysomes. After some preliminary mass spectrometry of polysomal fractions, we investigated the distributions of selected tagged proteins using sucrose gradients and immunofluorescence. As expected, the polysomal fractions contained nearly all annotated ribosomal proteins, the translation-associated protein folding complex, and many translation factors, but also many other abundant proteins. Results suggested that cap-binding proteins EIF4E3 and EIF4E4 were associated with both free and membrane-bound polysomes. The EIF4E binding partners EIF4G4 and EIF4G3 were present but the other EIF4E and EIF4G paralogues were not detected. The dominant EIF4E in the polysomal fraction is EIF4E4 and very few polysomal mRNAs are associated with EIF4G. Thirteen potential mRNA-binding proteins were detected in the polysomes, including the known polysome-associated protein RBP42. The locations of two of the other proteins were tested after epitope tagging: RBP29 was in the nucleus and ZC3H29 was in the cytoplasm. Quantitative analyses showed that specific association of an RNA-binding protein with the polysome fraction in sucrose gradients will not be detected if the protein is in more than 25-fold molar excess over its target binding sites. PMID:26287607

  19. Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy

    PubMed Central

    Nieuwkoop, Andrew J.; Berthold, Deborah A.; Comellas, Gemma; Sperling, Lindsay J.; Tang, Ming; Shah, Gautam J.; Brea, Elliott J.; Lemkau, Luisel R.

    2012-01-01

    Solid-state NMR has emerged as an important tool for structural biology and chemistry, capable of solving atomic-resolution structures for proteins in membrane-bound and aggregated states. Proton detection methods have been recently realized under fast magic-angle spinning conditions, providing large sensitivity enhancements for efficient examination of uniformly labeled proteins. The first and often most challenging step of protein structure determination by NMR is the site-specific resonance assignment. Here we demonstrate resonance assignments based on high-sensitivity proton-detected three-dimensional experiments for samples of different physical states, including a fully-protonated small protein (GB1, 6 kDa), a deuterated microcrystalline protein (DsbA, 21 kDa), a membrane protein (DsbB, 20 kDa) prepared in a lipid environment, and the extended core of a fibrillar protein (α-synuclein, 14 kDa). In our implementation of these experiments, including CONH, CO(CA)NH, CANH, CA(CO)NH, CBCANH, and CBCA(CO)NH, dipolar-based polarization transfer methods have been chosen for optimal efficiency for relatively high protonation levels (full protonation or 100 % amide proton), fast magic-angle spinning conditions (40 kHz) and moderate proton decoupling power levels. Each H–N pair correlates exclusively to either intra- or inter-residue carbons, but not both, to maximize spectral resolution. Experiment time can be reduced by at least a factor of 10 by using proton detection in comparison to carbon detection. These high-sensitivity experiments are especially important for membrane proteins, which often have rather low expression yield. Proton-detection based experiments are expected to play an important role in accelerating protein structure elucidation by solid-state NMR with the improved sensitivity and resolution. PMID:22986689

  20. Structure and protein-protein interactions of methanol dehydrogenase from Methylococcus capsulatus (Bath).

    PubMed

    Culpepper, Megen A; Rosenzweig, Amy C

    2014-10-01

    In the initial steps of their metabolic pathway, methanotrophic bacteria oxidize methane to methanol with methane monooxygenases (MMOs) and methanol to formaldehyde with methanol dehydrogenases (MDHs). Several lines of evidence suggest that the membrane-bound or particulate MMO (pMMO) and MDH interact to form a metabolic supercomplex. To further investigate the possible existence of such a supercomplex, native MDH from Methylococcus capsulatus (Bath) has been purified and characterized by size exclusion chromatography with multi-angle light scattering and X-ray crystallography. M. capsulatus (Bath) MDH is primarily a dimer in solution, although an oligomeric species with a molecular mass of ∼450-560 kDa forms at higher protein concentrations. The 2.57 Å resolution crystal structure reveals an overall fold and α2β2 dimeric architecture similar to those of other MDH structures. In addition, biolayer interferometry studies demonstrate specific protein-protein interactions between MDH and M. capsulatus (Bath) pMMO as well as between MDH and the truncated recombinant periplasmic domains of M. capsulatus (Bath) pMMO (spmoB). These interactions exhibit KD values of 833 ± 409 nM and 9.0 ± 7.7 μM, respectively. The biochemical data combined with analysis of the crystal lattice interactions observed in the MDH structure suggest a model in which MDH and pMMO associate not as a discrete, stoichiometric complex but as a larger assembly scaffolded by the intracytoplasmic membranes. PMID:25185034

  1. Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location.

    PubMed

    Liao, Shu Y; Lee, Myungwoon; Wang, Tuo; Sergeyev, Ivan V; Hong, Mei

    2016-03-01

    Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~fourfold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105-160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 Å from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes. PMID:26873390

  2. OPM database and PPM web server: resources for positioning of proteins in membranes

    PubMed Central

    Lomize, Mikhail A.; Pogozheva, Irina D.; Joo, Hyeon; Mosberg, Henry I.; Lomize, Andrei L.

    2012-01-01

    The Orientations of Proteins in Membranes (OPM) database is a curated web resource that provides spatial positions of membrane-bound peptides and proteins of known three-dimensional structure in the lipid bilayer, together with their structural classification, topology and intracellular localization. OPM currently contains more than 1200 transmembrane and peripheral proteins and peptides from approximately 350 organisms that represent approximately 3800 Protein Data Bank entries. Proteins are classified into classes, superfamilies and families and assigned to 21 distinct membrane types. Spatial positions of proteins with respect to the lipid bilayer are optimized by the PPM 2.0 method that accounts for the hydrophobic, hydrogen bonding and electrostatic interactions of the proteins with the anisotropic water-lipid environment described by the dielectric constant and hydrogen-bonding profiles. The OPM database is freely accessible at http://opm.phar.umich.edu. Data can be sorted, searched or retrieved using the hierarchical classification, source organism, localization in different types of membranes. The database offers downloadable coordinates of proteins and peptides with membrane boundaries. A gallery of protein images and several visualization tools are provided. The database is supplemented by the PPM server (http://opm.phar.umich.edu/server.php) which can be used for calculating spatial positions in membranes of newly determined proteins structures or theoretical models. PMID:21890895

  3. Heterogeneity of zein mRNA and protein in maize.

    PubMed

    Park, W D; Lewis, E D; Rubenstein, I

    1980-01-01

    Zein, the prolamine fraction of maize, is localized in the endosperm in membrane-bound structures called protein bodies, which have polyribosomes on their surfaces. These polysomes or the mRNA fraction isolated from them will direct the synthesis of zein-like proteins in vitro. The in vitro products consist primarily of two molecular weight classes but show considerable charge heterogeneity when analyzed by isoelectric focusing. Although the molecular weight classes are very similar for different inbred lines, the isoelectric focusing patterns differ.Results given here suggest that the extensive charge heterogeneity of zein proteins does not result from the presence of a large number of totally distinct mRNAs. Zein proteins synthesized in vitro fall into several families related by sequence homologies in their mRNAs. In Illinois High Protein (IHP) the major zein mRNAs can be classified into three families based on their binding to cloned complimentary DNA copies of IHP zein mRNA. Each of three other lines we have studied (W22, Oh43, and W64A) has zein mRNAs that are related to those of IHP. Among these four lines the molecular weights of the members of a given family are generally similar, but the number of members in a family and their isoelectric points differ. PMID:16661153

  4. Modulation of Rab GTPase function by a protein phosphocholine transferase.

    PubMed

    Mukherjee, Shaeri; Liu, Xiaoyun; Arasaki, Kohei; McDonough, Justin; Galán, Jorge E; Roy, Craig R

    2011-09-01

    The intracellular pathogen Legionella pneumophila modulates the activity of host GTPases to direct the transport and assembly of the membrane-bound compartment in which it resides. In vitro studies have indicated that the Legionella protein DrrA post-translationally modifies the GTPase Rab1 by a process called AMPylation. Here we used mass spectrometry to investigate post-translational modifications to Rab1 that occur during infection of host cells by Legionella. Consistent with in vitro studies, DrrA-mediated AMPylation of a conserved tyrosine residue in the switch II region of Rab1 was detected during infection. In addition, a modification to an adjacent serine residue in Rab1 was discovered, which was independent of DrrA. The Legionella effector protein AnkX was required for this modification. Biochemical studies determined that AnkX directly mediates the covalent attachment of a phosphocholine moiety to Rab1. This phosphocholine transferase activity used CDP-choline as a substrate and required a conserved histidine residue located in the FIC domain of the AnkX protein. During infection, AnkX modified both Rab1 and Rab35, which explains how this protein modulates membrane transport through both the endocytic and exocytic pathways of the host cell. Thus, phosphocholination of Rab GTPases represents a mechanism by which bacterial FIC-domain-containing proteins can alter host-cell functions. PMID:21822290

  5. Investigation of molecular mechanisms of action of chelating drugs on protein-lipid model membranes by X-ray fluorescence

    SciTech Connect

    Novikova, N. N.; Zheludeva, S. I.; Koval'chuk, M. V.; Stepina, N. D.; Erko, A. I.; Yur'eva, E. A.

    2009-12-15

    Protein-lipid films based on the enzyme alkaline phosphatase were subjected to the action of chelating drugs, which are used for accelerating the removal of heavy metals from the human body, and the elemental composition of the resulting films was investigated. Total-reflection X-ray fluorescence measurements were performed at the Berlin Electron Storage Ring Company for Synchrotron Radiation (BESSY) in Germany. A comparative estimation of the protective effect of four drugs (EDTA, succimer, xydiphone, and mediphon) on membrane-bound enzymes damaged by lead ions was made. The changes in the elemental composition of the protein-lipid films caused by high doses of chelating drugs were investigated. It was shown that state-of-the-art X-ray techniques can, in principle, be used to develop new methods for the in vitro evaluation of the efficiency of drugs, providing differential data on their actions.

  6. The intact Kunitz domain protects the amyloid precursor protein from being processed by matriptase-2.

    PubMed

    Beckmann, Anna-Madeleine; Glebov, Konstantin; Walter, Jochen; Merkel, Olaf; Mangold, Martin; Schmidt, Frederike; Becker-Pauly, Christoph; Gütschow, Michael; Stirnberg, Marit

    2016-08-01

    Proteolytic processing of the amyloid precursor protein (APP) leads to amyloid-β (Aβ) peptides. So far, the mechanism of APP processing is insufficiently characterized at the molecular level. Whereas the knowledge of Aβ generation by several proteases has been expanded, the contribution of the Kunitz-type protease inhibitor domain (KPI) present in two major APP isoforms to the complex proteolytic processing of APP is poorly understood. In this study, we have identified KPI-containing APP as a very potent, slow-binding inhibitor for the membrane-bound proteolytic regulator of iron homeostasis matriptase-2 by forming stable complexes with its target protease in HEK cells. Inhibition and complex formation depend on the intact KPI domain. By inhibiting matriptase-2, KPI-containing APP is protected from matriptase-2-mediated proteolysis within the Aβ region, thus preventing the generation of N-terminally truncated Aβ. PMID:27078672

  7. A consensus procedure for predicting the location of alpha-helical transmembrane segments in proteins.

    PubMed

    Parodi, L A; Granatir, C A; Maggiora, G M

    1994-09-01

    To aid in the development of three-dimensional models of membrane-bound proteins, a consensus procedure for predicting alpha-helical transmembrane segments from amino acid sequence is presented. The algorithm combines the results of six individual prediction methods and some basic properties of membrane-spanning helices to obtain a final consensus prediction. Comparison with experiment and several other recently developed methods shows that the consensus procedure performs quite well in comparison to other recent methods. A FORTRAN program has been developed which takes an input file containing an amino acid sequence in one-letter code and outputs a list of the alpha-helical transmembrane segments predicted by the consensus algorithm. PMID:7828069

  8. The novel protein Ccz1p required for vacuolar assembly in Saccharomyces cerevisiae functions in the same transport pathway as Ypt7p.

    PubMed

    Kucharczyk, R; Dupre, S; Avaro, S; Haguenauer-Tsapis, R; Słonimski, P P; Rytka, J

    2000-12-01

    CCZ1 was previously identified by the sensitivity of ccz1(delta) mutants to high concentrations of Caffeine and the divalent ions Ca(2+ )and Zn(2+). In this paper we show that deletion of CCZ1 leads to aberrant vacuole morphology, similar to the one reported for the family of vacuolar protein sorting (vps) mutants of class B. The ccz1(&Dgr;) cells display severe vacuolar protein sorting defects for both the soluble carboxipeptidase Y and the membrane-bound alkaline phosphatase, which are delivered to the vacuole by distinct routes. Ccz1p is a membranous protein and the vast majority of Ccz1p resides in late endosomes. These results, along with a functional linkage found between the CCZ1 and YPT7 genes, indicate that the site of Ccz1p function is at the last step of fusion of multiple transport intermediates with the vacuole. PMID:11069774

  9. Application of a free-energy-landscape approach to study tension-dependent bilayer tubulation mediated by curvature-inducing proteins

    NASA Astrophysics Data System (ADS)

    Tourdot, Richard W.; Ramakrishnan, N.; Baumgart, Tobias; Radhakrishnan, Ravi

    2015-10-01

    We investigate the phenomenon of protein-induced tubulation of lipid bilayer membranes within a continuum framework using Monte Carlo simulations coupled with the Widom insertion technique to compute excess chemical potentials. Tubular morphologies are spontaneously formed when the density and the curvature-field strength of the membrane-bound proteins exceed their respective thresholds and this transition is marked by a sharp drop in the excess chemical potential. We find that the planar to tubular transition can be described by a micellar model and that the corresponding free-energy barrier increases with an increase in the curvature-field strength (i.e., of protein-membrane interactions) and also with an increase in membrane tension.

  10. Application of a free-energy-landscape approach to study tension-dependent bilayer tubulation mediated by curvature-inducing proteins.

    PubMed

    Tourdot, Richard W; Ramakrishnan, N; Baumgart, Tobias; Radhakrishnan, Ravi

    2015-10-01

    We investigate the phenomenon of protein-induced tubulation of lipid bilayer membranes within a continuum framework using Monte Carlo simulations coupled with the Widom insertion technique to compute excess chemical potentials. Tubular morphologies are spontaneously formed when the density and the curvature-field strength of the membrane-bound proteins exceed their respective thresholds and this transition is marked by a sharp drop in the excess chemical potential. We find that the planar to tubular transition can be described by a micellar model and that the corresponding free-energy barrier increases with an increase in the curvature-field strength (i.e., of protein-membrane interactions) and also with an increase in membrane tension. PMID:26565280

  11. Application of a free-energy-landscape approach to study tension-dependent bilayer tubulation mediated by curvature-inducing proteins

    PubMed Central

    Tourdot, Richard W.; Ramakrishnan, N.; Baumgart, Tobias; Radhakrishnan, Ravi

    2016-01-01

    We investigate the phenomenon of protein-induced tubulation of lipid bilayer membranes within a continuum framework using Monte Carlo simulations coupled with the Widom insertion technique to compute excess chemical potentials. Tubular morphologies are spontaneously formed when the density and the curvature-field strength of the membrane-bound proteins exceed their respective thresholds and this transition is marked by a sharp drop in the excess chemical potential. We find that the planar to tubular transition can be described by a micellar model and that the corresponding free-energy barrier increases with an increase in the curvature-field strength (i.e., of protein-membrane interactions) and also with an increase in membrane tension. PMID:26565280

  12. The 1.35 A resolution structure of the phosphatase domain of the suppressor of T cell receptor signaling protein in complex with sulfate

    SciTech Connect

    Jakoncic, J.; Sondgeroth, B.; Carpino, N.; Nassar, N.

    2010-04-19

    The suppressor of T-cell signaling (Sts) proteins are multidomain proteins that negatively regulate the signaling of membrane-bound receptors, including the T-cell receptor (TCR) and the epidermal growth-factor receptor (EGFR). They contain at their C-terminus a 2H-phosphatase homology (PGM) domain that is responsible for their protein tyrosine phosphatase activity. Here, the crystal structure of the phosphatase domain of Sts-1, Sts-1PGM, was determined at pH 4.6. The asymmetric unit contains two independent molecules and each active site is occupied by a sulfate ion. Each sulfate is located at the phosphate-binding site and makes similar interactions with the catalytic residues. The structure suggests an explanation for the lower Michaelis-Menten constants at acidic pH.

  13. Proteomic identification of dysferlin-interacting protein complexes in human vascular endothelium

    SciTech Connect

    Leung, Cleo; Utokaparch, Soraya; Sharma, Arpeeta; Yu, Carol; Abraham, Thomas; Borchers, Christoph; Bernatchez, Pascal

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Bi-directional (inward and outward) movement of GFP-dysferlin in COS-7 cells. Black-Right-Pointing-Pointer Dysferlin interacts with key signaling proteins for transcytosis in EC. Black-Right-Pointing-Pointer Dysferlin mediates trafficking of vesicles carrying protein cargos in EC. -- Abstract: Dysferlin is a membrane-anchored protein known to facilitate membrane repair in skeletal muscles following mechanical injury. Mutations of dysferlin gene impair sarcolemma integrity, a hallmark of certain forms of muscular dystrophy in patients. Dysferlin contains seven calcium-dependent C2 binding domains, which are required to promote fusion of intracellular membrane vesicles. Emerging evidence reveal the unexpected expression of dysferlin in non-muscle, non-mechanically active tissues, such as endothelial cells, which cast doubts over the belief that ferlin proteins act exclusively as membrane repair proteins. We and others have shown that deficient trafficking of membrane bound proteins in dysferlin-deficient cells, suggesting that dysferlin might mediate trafficking of client proteins. Herein, we describe the intracellular trafficking and movement of GFP-dysferlin positive vesicles in unfixed reconstituted cells using live microscopy. By performing GST pull-down assays followed by mass spectrometry, we identified dysferlin binding protein complexes in human vascular endothelial cells. Together, our data further support the claims that dysferlin not only mediates membrane repair but also trafficking of client proteins, ultimately, help bridging dysferlinopathies to aberrant membrane signaling.

  14. Fatty acylation of proteins: The long and the short of it.

    PubMed

    Resh, Marilyn D

    2016-07-01

    Long, short and medium chain fatty acids are covalently attached to hundreds of proteins. Each fatty acid confers distinct biochemical properties, enabling fatty acylation to regulate intracellular trafficking, subcellular localization, protein-protein and protein-lipid interactions. Myristate and palmitate represent the most common fatty acid modifying groups. New insights into how fatty acylation reactions are catalyzed, and how fatty acylation regulates protein structure and function continue to emerge. Myristate is typically linked to an N-terminal glycine, but recent studies reveal that lysines can also be myristoylated. Enzymes that remove N-terminal myristoyl-glycine or myristate from lysines have now been identified. DHHC proteins catalyze S-palmitoylation, but the mechanisms that regulate substrate recognition by individual DHHC family members remain to be determined. New studies continue to reveal thioesterases that remove palmitate from S-acylated proteins. Another area of rapid expansion is fatty acylation of the secreted proteins hedgehog, Wnt and Ghrelin, by Hhat, Porcupine and GOAT, respectively. Understanding how these membrane bound O-acyl transferases recognize their protein and fatty acyl CoA substrates is an active area of investigation, and is punctuated by the finding that these enzymes are potential drug targets in human diseases. PMID:27233110

  15. Identification of guanylate cyclases and related signaling proteins in sperm tail from sea stars by mass spectrometry.

    PubMed

    Nakachi, Mia; Matsumoto, Midori; Terry, Philip M; Cerny, Ronald L; Moriyama, Hideaki

    2008-01-01

    Marine invertebrates employ external fertilization to take the advantages of sexual reproduction as one of excellent survival strategies. To prevent mismatching, successful fertilization can be made only after going though strictly defined steps in the fertilization. In sea stars, the fertilization process starts with the chemotaxis of sperm followed by hyperactivation of sperm upon arriving onto the egg coat, and then sperm penetrate to the egg coat before achieving the fusion. To investigate whether the initiation of chemotaxis and the following signaling has species specificity, we conducted comparative studies in the protein level among sea stars, Asterias amurensis, A. forbesi, and Asterina pectinifera. Since transcription of messenger ribonucleic acid (mRNA) has been suppressed in gamete, the roles of sperm proteins during the fertilization cannot be investigated by examining the mRNA profile. Therefore, proteomics analysis by mass spectrometry was used in this study. In sea stars, upon receiving asteroidal sperm-activating peptide (asterosap), the receptor membrane-bound guanylate cyclases in the sperm tail trigger sperm chemotaxis. We confirmed the presence of membrane-bound guanylate cyclases in the three sea star species, and they all had the same structural domains including the extracellular domain, kinase-like domain, and guanylate cyclase domain. The majority of peptides recovered were from alpha-helices distributed on the solvent side of the protein. More peptides were recovered from the intracellular domains. The transmembrane domain has not been recovered. The functions of the receptors seemed to be conserved among the species. Furthermore, we identified proteins that may be involved in the guanylate cyclase-triggered signaling pathway. PMID:18461395

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-05-01

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

  18. Membrane-binding properties of phospholipase C-beta1 and phospholipaseC-beta2: role of the C-terminus and effects of polyphosphoinositides, G-proteins and Ca2+.

    PubMed Central

    Jenco, J M; Becker, K P; Morris, A J

    1997-01-01

    We have studied the binding of two G-protein-regulated phospholipase C (PLC) enzymes, PLCs-beta1 and -beta2, to membrane surfaces using sucrose-loaded bilayer phospholipid vesicles of varying compositions. Neither enzyme binds appreciably to pure phosphatidylcholine vesicles at lipid concentrations up to 10(-3) M. PLC-beta1 and PLC-beta2 bind vesicles composed of phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine (molar ratio 1:1:1) with an approximate Kd of 10(-5) M. Inclusion of 2% PtdIns(4,5)P2 in these vesicles had no effect on the affinity of this interaction. As reported by others, removal of the C-terminus of PLC-beta1 and PLC-beta2 produces catalytically active fragments. The affinity of these truncated proteins for phospholipid vesicles is dramatically reduced suggesting that this region of the proteins contains residues important for membrane binding. Inclusion of G-protein alpha- and betagamma-subunit activators in the phospholipid vesicles does not increase the binding of PLC-beta1 or PLC-beta2, and the magnitude of G-protein-mediated PLC activation observed at low phospholipid concentrations (10(-6) M) is comparable to that observed at concentrations at which the enzymes are predominantly membrane-bound (10(-3) M). PLC-beta1 and -beta2 contain C2 domains but Ca2+ does not enhance binding to the vesicles. Our results indicate that binding of these enzymes to membranes involves the C-temini of the proteins and suggest that activation of these enzymes by G-proteins results from a regulated interaction between the membrane-bound proteins rather than G-protein-dependent recruitment of soluble enzymes to a substrate-containing phospholipid surface. PMID:9359412

  19. Heterodimeric Capping Protein from Arabidopsis Is a Membrane-Associated, Actin-Binding Protein1[W][OPEN

    PubMed Central

    Jimenez-Lopez, Jose C.; Wang, Xia; Kotchoni, Simeon O.; Huang, Shanjin; Szymanski, Daniel B.; Staiger, Christopher J.

    2014-01-01

    The actin cytoskeleton is a major regulator of cell morphogenesis and responses to biotic and abiotic stimuli. The organization and activities of the cytoskeleton are choreographed by hundreds of accessory proteins. Many actin-binding proteins are thought to be stimulus-response regulators that bind to signaling phospholipids and change their activity upon lipid binding. Whether these proteins associate with and/or are regulated by signaling lipids in plant cells remains poorly understood. Heterodimeric capping protein (CP) is a conserved and ubiquitous regulator of actin dynamics. It binds to the barbed end of filaments with high affinity and modulates filament assembly and disassembly reactions in vitro. Direct interaction of CP with phospholipids, including phosphatidic acid, results in uncapping of filament ends in vitro. Live-cell imaging and reverse-genetic analyses of cp mutants in Arabidopsis (Arabidopsis thaliana) recently provided compelling support for a model in which CP activity is negatively regulated by phosphatidic acid in vivo. Here, we used complementary biochemical, subcellular fractionation, and immunofluorescence microscopy approaches to elucidate CP-membrane association. We found that CP is moderately abundant in Arabidopsis tissues and present in a microsomal membrane fraction. Sucrose density gradient separation and immunoblotting with known compartment markers were used to demonstrate that CP is enriched on membrane-bound organelles such as the endoplasmic reticulum and Golgi. This association could facilitate cross talk between the actin cytoskeleton and a wide spectrum of essential cellular functions such as organelle motility and signal transduction. PMID:25201878

  20. The similarity between N-terminal targeting signals for protein import into different organelles and its evolutionary relevance

    PubMed Central

    Kunze, Markus; Berger, Johannes

    2015-01-01

    The proper distribution of proteins between the cytosol and various membrane-bound compartments is crucial for the functionality of eukaryotic cells. This requires the cooperation between protein transport machineries that translocate diverse proteins from the cytosol into these compartments and targeting signal(s) encoded within the primary sequence of these proteins that define their cellular destination. The mechanisms exerting protein translocation differ remarkably between the compartments, but the predominant targeting signals for mitochondria, chloroplasts and the ER share the N-terminal position, an α-helical structural element and the removal from the core protein by intraorganellar cleavage. Interestingly, similar properties have been described for the peroxisomal targeting signal type 2 mediating the import of a fraction of soluble peroxisomal proteins, whereas other peroxisomal matrix proteins encode the type 1 targeting signal residing at the extreme C-terminus. The structural similarity of N-terminal targeting signals poses a challenge to the specificity of protein transport, but allows the generation of ambiguous targeting signals that mediate dual targeting of proteins into different compartments. Dual targeting might represent an advantage for adaptation processes that involve a redistribution of proteins, because it circumvents the hierarchy of targeting signals. Thus, the co-existence of two equally functional import pathways into peroxisomes might reflect a balance between evolutionary constant and flexible transport routes. PMID:26441678

  1. Chronic hypoxia-induced alteration of presynaptic protein profiles and neurobehavioral dysfunction are averted by supplemental oxygen in Lymnaea stagnalis.

    PubMed

    Fei, G-H; Feng, Z-P

    2008-04-22

    Chronic hypoxia causes neural dysfunction. Oxygen (O(2)) supplements have been commonly used to increase the O(2) supply, yet the therapeutic benefit of this treatment remains controversial due to a lack of cellular and molecular evidence. In this study, we examined the effects of short-burst O(2) supplementation on neural behavior and presynaptic protein expression profiles in a simple chronic hypoxia model of snail Lymnaea stagnalis. We reported that hypoxia delayed the animal response to light stimuli, suppressed locomotory activity, induced expression of stress-response proteins, hypoxia inducible factor-1alpha (HIF-1alpha) and heat shock protein 70 (HSP70), repressed syntaxin-1 (a membrane-bound presynaptic protein) and elevated vesicle-associated membrane protein-1 (VAMP-1) (a vesicle-bound presynaptic protein) level. O(2) supplements relieved suppression of neural behaviors, and corrected hypoxia-induced protein alterations in a dose-dependent manner. The effectiveness of supplemental O(2) was further evaluated by determining time courses for recovery of neural behaviors and expression of stress response proteins and presynaptic proteins after relief from hypoxia conditions. Our findings suggest that O(2) supplement improves hypoxia-induced adverse alterations of presynaptic protein expression and neurobehaviors, however, the optimal level of O(2) required for improvement is protein specific and system specific. PMID:18343591

  2. Microcompartments and Protein Machines in Prokaryotes

    PubMed Central

    Saier, Milton H.

    2013-01-01

    The prokaryotic cell was once thought of as a “bag of enzymes” with little or no intracellular compartmentalization. In this view, most reactions essential for life occurred as a consequence of random molecular collisions involving substrates, cofactors and cytoplasmic enzymes. Our current conception of a prokaryote is far from this view. We now consider a bacterium or an archaeon as a highly structured, non-random collection of functional membrane-embedded and proteinaceous molecular machines, each of which serves a specialized function. In this article we shall present an overview of such microcompartments including (i) the bacterial cytoskeleton and the apparati allowing DNA segregation during cells division, (ii) energy transduction apparati involving light-driven proton pumping and ion gradient-driven ATP synthesis, (iii) prokaryotic motility and taxis machines that mediate cell movements in response to gradients of chemicals and physical forces, (iv) machines of protein folding, secretion and degradation, (v) metabolasomes carrying out specific chemical reactions, (vi) 24 hour clocks allowing bacteria to coordinate their metabolic activities with the daily solar cycle and (vii) proteinaceous membrane compartmentalized structures such as sulfur granules and gas vacuoles. Membrane-bounded prokaryotic organelles were considered in a recent JMMB written symposium concerned with membraneous compartmentalization in bacteria [Saier and Bogdanov, 2013]. By contrast, in this symposium, we focus on proteinaceous microcompartments. These two symposia, taken together, provide the interested reader with an objective view of the remarkable complexity of what was once thought of as a simple non-compartmentalized cell. PMID:23920489

  3. Prediction of protein orientation upon immobilization on biological and nonbiological surfaces.

    PubMed

    Talasaz, AmirAli H; Nemat-Gorgani, Mohsen; Liu, Yang; Ståhl, Patrik; Dutton, Robert W; Ronaghi, Mostafa; Davis, Ronald W

    2006-10-01

    We report on a rapid simulation method for predicting protein orientation on a surface based on electrostatic interactions. New methods for predicting protein immobilization are needed because of the increasing use of biosensors and protein microarrays, two technologies that use protein immobilization onto a solid support, and because the orientation of an immobilized protein is important for its function. The proposed simulation model is based on the premise that the protein interacts with the electric field generated by the surface, and this interaction defines the orientation of attachment. Results of this model are in agreement with experimental observations of immobilization of mitochondrial creatine kinase and type I hexokinase on biological membranes. The advantages of our method are that it can be applied to any protein with a known structure; it does not require modeling of the surface at atomic resolution and can be run relatively quickly on readily available computing resources. Finally, we also propose an orientation of membrane-bound cytochrome c, a protein for which the membrane orientation has not been unequivocally determined. PMID:17001006

  4. A subset of bacterial inner membrane proteins integrated by the twin-arginine translocase.

    PubMed

    Hatzixanthis, Kostas; Palmer, Tracy; Sargent, Frank

    2003-09-01

    A group of bacterial exported proteins are synthesized with N-terminal signal peptides containing a SRRxFLK 'twin-arginine' amino acid motif. Proteins bearing twin-arginine signal peptides are targeted post-translationally to the twin-arginine translocation (Tat) system which transports folded substrates across the inner membrane. In Escherichia coli, most integral inner membrane proteins are assembled by a co-translational process directed by SRP/FtsY, the SecYEG translocase, and YidC. In this work we define a novel class of integral membrane proteins assembled by a Tat-dependent mechanism. We show that at least five E. coli Tat substrate proteins contain hydrophobic C-terminal transmembrane helices (or 'C-tails'). Fusions between the identified transmembrane C-tails and the exclusively Tat-dependent reporter proteins TorA and SufI render the resultant chimeras membrane-bound. Export-linked signal peptide processing and membrane integration of the chimeras is shown to be both Tat-dependent and YidC-independent. It is proposed that the mechanism of membrane integration of proteins by the Tat system is fundamentally distinct from that employed for other bacterial inner membrane proteins. PMID:12940994

  5. Prediction of protein orientation upon immobilization on biological and nonbiological surfaces

    NASA Astrophysics Data System (ADS)

    Talasaz, Amirali H.; Nemat-Gorgani, Mohsen; Liu, Yang; Ståhl, Patrik; Dutton, Robert W.; Ronaghi, Mostafa; Davis, Ronald W.

    2006-10-01

    We report on a rapid simulation method for predicting protein orientation on a surface based on electrostatic interactions. New methods for predicting protein immobilization are needed because of the increasing use of biosensors and protein microarrays, two technologies that use protein immobilization onto a solid support, and because the orientation of an immobilized protein is important for its function. The proposed simulation model is based on the premise that the protein interacts with the electric field generated by the surface, and this interaction defines the orientation of attachment. Results of this model are in agreement with experimental observations of immobilization of mitochondrial creatine kinase and type I hexokinase on biological membranes. The advantages of our method are that it can be applied to any protein with a known structure; it does not require modeling of the surface at atomic resolution and can be run relatively quickly on readily available computing resources. Finally, we also propose an orientation of membrane-bound cytochrome c, a protein for which the membrane orientation has not been unequivocally determined. electric double layer | electrostatic simulations | orientation flexibility

  6. A Systematic Cell-Based Analysis of Localization of Predicted Drosophila Peroxisomal Proteins.

    PubMed

    Baron, Matthew N; Klinger, Christen M; Rachubinski, Richard A; Simmonds, Andrew J

    2016-05-01

    Peroxisomes are membrane-bound organelles found in almost all eukaryotic cells. They perform specialized biochemical functions that vary with organism, tissue or cell type. Mutations in human genes required for the assembly of peroxisomes result in a spectrum of diseases called the peroxisome biogenesis disorders. A previous sequence-based comparison of the predicted proteome of Drosophila melanogaster (the fruit fly) to human proteins identified 82 potential homologues of proteins involved in peroxisomal biogenesis, homeostasis or metabolism. However, the subcellular localization of these proteins relative to the peroxisome was not determined. Accordingly, we tested systematically the localization and selected functions of epitope-tagged proteins in Drosophila Schneider 2 cells to determine the subcellular localization of 82 potential Drosophila peroxisomal protein homologues. Excluding the Pex proteins, 34 proteins localized primarily to the peroxisome, 8 showed dual localization to the peroxisome and other structures, and 26 localized exclusively to organelles other than the peroxisome. Drosophila is a well-developed laboratory animal often used for discovery of gene pathways, including those linked to human disease. Our work establishes a basic understanding of peroxisome protein localization in Drosophila. This will facilitate use of Drosophila as a genetically tractable, multicellular model system for studying key aspects of human peroxisome disease. PMID:26865094

  7. Mapping of the Regions Involved in Homotypic Interactions of Tula Hantavirus N Protein

    PubMed Central

    Kaukinen, Pasi; Vaheri, Antti; Plyusnin, Alexander

    2003-01-01

    Hantavirus nucleocapsid (N) protein has been suggested to form homodimers and homotrimers that are further integrated into the nucleocapsid filaments around the viral RNA. Here we report detailed mapping of the regions involved in the homotypic N protein interactions in Tula hantavirus (TULV). Peptide scan screening was used to define the interaction regions, and the mammalian two-hybrid assay was used for the functional analysis of N protein mutants. To study linear regions responsible for N protein interaction(s), we used peptide scanning in which N peptides synthesized on membranes recognize recombinant TULV N protein. The data showed that the N protein bound to membrane-bound peptides comprising amino acids 13 to 30 and 41 to 57 in the N-terminal part and 340 to 379, 391 to 407, and 410 to 419 in the C-terminal part of the molecule. Further mapping of the interaction regions by alanine scanning indicated the importance of basic amino acids along the N protein and especially asparagine-394, histidine-395, and phenyalanine-396 in forming the binding interface. Analysis of truncated mutants in the mammalian two-hybrid assay showed that N-terminal amino acids 1 to 43 are involved in and C-terminal amino acids 393 to 398 (VNHFHL) are absolutely crucial for the homotypic interactions. Furthermore, our data suggested a tail-to-tail and head-to-head binding scheme for the N proteins. PMID:14512541

  8. The invariant phenylalanine of precursor proteins discloses the importance of Omp85 for protein translocation into cyanelles

    PubMed Central

    Wunder, Tobias; Martin, Roman; Löffelhardt, Wolfgang; Schleiff, Enrico; Steiner, Jürgen M

    2007-01-01

    Background Today it is widely accepted that plastids are of cyanobacterial origin. During their evolutionary integration into the metabolic and regulatory networks of the host cell the engulfed cyanobacteria lost their independency. This process was paralleled by a massive gene transfer from symbiont to the host nucleus challenging the development of a retrograde protein translocation system to ensure plastid functionality. Such a system includes specific targeting signals of the proteins needed for the function of the plastid and membrane-bound machineries performing the transfer of these proteins across the envelope membranes. At present, most information on protein translocation is obtained by the analysis of land plants. However, the analysis of protein import into the primitive plastids of glaucocystophyte algae, revealed distinct features placing this system as a tool to understand the evolutionary development of translocation systems. Here, bacterial outer membrane proteins of the Omp85 family have recently been discussed as evolutionary seeds for the development of translocation systems. Results To further explore the initial mode of protein translocation, the observed phenylalanine dependence for protein translocation into glaucophyte plastids was pursued in detail. We document that indeed the phenylalanine has an impact on both, lipid binding and binding to proteoliposomes hosting an Omp85 homologue. Comparison to established import experiments, however, unveiled a major importance of the phenylalanine for recognition by Omp85. This finding is placed into the context of the evolutionary development of the plastid translocon. Conclusion The phenylalanine in the N-terminal domain signs as a prerequisite for protein translocation across the outer membrane assisted by a "primitive" translocon. This amino acid appears to be optimized for specifically targeting the Omp85 protein without enforcing aggregation on the membrane surface. The phenylalanine has

  9. Eps15 Homology Domain-containing Protein 3 Regulates Cardiac T-type Ca2+ Channel Targeting and Function in the Atria.

    PubMed

    Curran, Jerry; Musa, Hassan; Kline, Crystal F; Makara, Michael A; Little, Sean C; Higgins, John D; Hund, Thomas J; Band, Hamid; Mohler, Peter J

    2015-05-01

    Proper trafficking of membrane-bound ion channels and transporters is requisite for normal cardiac function. Endosome-based protein trafficking of membrane-bound ion channels and transporters in the heart is poorly understood, particularly in vivo. In fact, for select cardiac cell types such as atrial myocytes, virtually nothing is known regarding endosomal transport. We previously linked the C-terminal Eps15 homology domain-containing protein 3 (EHD3) with endosome-based protein trafficking in ventricular cardiomyocytes. Here we sought to define the roles and membrane protein targets for EHD3 in atria. We identify the voltage-gated T-type Ca(2+) channels (CaV3.1, CaV3.2) as substrates for EHD3-dependent trafficking in atria. Mice selectively lacking EHD3 in heart display reduced expression and targeting of both Cav3.1 and CaV3.2 in the atria. Furthermore, functional experiments identify a significant loss of T-type-mediated Ca(2+) current in EHD3-deficient atrial myocytes. Moreover, EHD3 associates with both CaV3.1 and CaV3.2 in co-immunoprecipitation experiments. T-type Ca(2+) channel function is critical for proper electrical conduction through the atria. Consistent with these roles, EHD3-deficient mice demonstrate heart rate variability, sinus pause, and atrioventricular conduction block. In summary, our findings identify CaV3.1 and CaV3.2 as substrates for EHD3-dependent protein trafficking in heart, provide in vivo data on endosome-based trafficking pathways in atria, and implicate EHD3 as a key player in the regulation of atrial myocyte excitability and cardiac conduction. PMID:25825486

  10. Eps15 Homology Domain-containing Protein 3 Regulates Cardiac T-type Ca2+ Channel Targeting and Function in the Atria*

    PubMed Central

    Curran, Jerry; Musa, Hassan; Kline, Crystal F.; Makara, Michael A.; Little, Sean C.; Higgins, John D.; Hund, Thomas J.; Band, Hamid; Mohler, Peter J.

    2015-01-01

    Proper trafficking of membrane-bound ion channels and transporters is requisite for normal cardiac function. Endosome-based protein trafficking of membrane-bound ion channels and transporters in the heart is poorly understood, particularly in vivo. In fact, for select cardiac cell types such as atrial myocytes, virtually nothing is known regarding endosomal transport. We previously linked the C-terminal Eps15 homology domain-containing protein 3 (EHD3) with endosome-based protein trafficking in ventricular cardiomyocytes. Here we sought to define the roles and membrane protein targets for EHD3 in atria. We identify the voltage-gated T-type Ca2+ channels (CaV3.1, CaV3.2) as substrates for EHD3-dependent trafficking in atria. Mice selectively lacking EHD3 in heart display reduced expression and targeting of both Cav3.1 and CaV3.2 in the atria. Furthermore, functional experiments identify a significant loss of T-type-mediated Ca2+ current in EHD3-deficient atrial myocytes. Moreover, EHD3 associates with both CaV3.1 and CaV3.2 in co-immunoprecipitation experiments. T-type Ca2+ channel function is critical for proper electrical conduction through the atria. Consistent with these roles, EHD3-deficient mice demonstrate heart rate variability, sinus pause, and atrioventricular conduction block. In summary, our findings identify CaV3.1 and CaV3.2 as substrates for EHD3-dependent protein trafficking in heart, provide in vivo data on endosome-based trafficking pathways in atria, and implicate EHD3 as a key player in the regulation of atrial myocyte excitability and cardiac conduction. PMID:25825486

  11. Cardiac muscle cell cytoskeletal protein 4.1: analysis of transcripts and subcellular location--relevance to membrane integrity, microstructure, and possible role in heart failure.

    PubMed

    Taylor-Harris, Pamela M; Keating, Lisa A; Maggs, Alison M; Phillips, Gareth W; Birks, Emma J; Franklin, Rodney C G; Yacoub, Magdi H; Baines, Anthony J; Pinder, Jennifer C

    2005-03-01

    The spectrin-based cytoskeleton assembly has emerged as a major player in heart functioning; however, cardiac protein 4.1, a key constituent, is uncharacterized. Protein 4.1 evolved to protect cell membranes against mechanical stresses and to organize membrane microstructure. 4.1 Proteins are multifunctional and, among other activities, link integral/signaling proteins on the plasma and internal membranes with the spectrin-based cytoskeleton. Four genes, EPB41, EPB41L1, EPB41L2, and EPB41L3 encode proteins 4.1R, 4.1N, 4.1G, and 4.1B, respectively. All are extensively spliced. Different isoforms are expressed according to tissue and developmental state, individual function being controlled through inclusion/exclusion of interactive domains. We have defined mouse and human cardiac 4.1 transcripts; other than 4. 1B in humans, all genes show activity. Cardiac transcripts constitutively include conserved FERM and C-terminal domains; both interact with membrane-bound signaling/transport/cell adhesion molecules. Variable splicing within and adjacent to the central spectrin/actin-binding domain enables regulation of cytoskeleton-binding activity. A novel heart-specific exon occurs in human 4.1G, but not in mouse. Immunofluorescence reveals 4.1 staining within mouse cardiomyocytes; thus, both at the plasma membrane and, interdigitated with sarcomeric myosin, across myofibrils in regions close to the sarcoplasmic reticulum. These are all regions to which spectrin locates. 4.1R in human heart shows similar distribution; however, there is limited plasma membrane staining. We conclude that cardiac 4.1s are highly regulated in their ability to crosslink plasma/integral cell membranes with the spectrin-actin cytoskeleton. We speculate that over the repetitive cycles of heart muscle contraction and relaxation, 4.1s are likely to locate, support, and coordinate functioning of key membrane-bound macromolecular assemblies. PMID:15834631

  12. Structural studies of human glioma pathogenesis-related protein 1

    SciTech Connect

    Asojo, Oluwatoyin A.; Koski, Raymond A.; Bonafé, Nathalie

    2011-10-01

    Structural analysis of a truncated soluble domain of human glioma pathogenesis-related protein 1, a membrane protein implicated in the proliferation of aggressive brain cancer, is presented. Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but is barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide that directs its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins) domain and a transmembrane domain. GLIPR1 is currently being investigated as a candidate for prostate cancer gene therapy and for glioblastoma targeted therapy. Crystal structures of a truncated soluble domain of the human GLIPR1 protein (sGLIPR1) solved by molecular replacement using a truncated polyalanine search model of the CAP domain of stecrisp, a snake-venom cysteine-rich secretory protein (CRISP), are presented. The correct molecular-replacement solution could only be obtained by removing all loops from the search model. The native structure was refined to 1.85 Å resolution and that of a Zn{sup 2+} complex was refined to 2.2 Å resolution. The latter structure revealed that the putative binding cavity coordinates Zn{sup 2+} similarly to snake-venom CRISPs, which are involved in Zn{sup 2+}-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop/turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. A model is also proposed for the structure of full-length membrane-bound GLIPR1.

  13. Growth hormone receptor/binding protein: Physiology and function

    SciTech Connect

    Herington, A.C.; Ymer, S.I.; Stevenson, J.L.; Roupas, P.

    1994-12-31

    Soluble truncated forms of the growth hormone receptor (GHR) are present in the circulation of many species and are also produced by many tissues/cell types. The major high-affinity forms of these GH-binding proteins (GHBP) are derived by alternative splicing of GHR mRNA in rodents, but probably by proteolytic cleavage in other species. Questions still remain with respect to the origins, native molecular forms(s), physiology, and function of the GHBPs, however. The observation that GH induces dimerization of the soluble GHBP and a membrane GHR, and that dimerization of GHR appears to be critical for GH bioactivity suggests that the presentation of GH to target cells, in an unbound form or as a monomeric or dimeric complex with GHBP, may have significant implications for the ability of GH to activate specific postreceptor signaling pathways (tyrosine kinase, protein kinase C, G-protein pathways) known to be utilized by GH for its diverse biological effects. This minireview addresses some of these aspects and highlights several new questions which have arisen as a result of recent advances in our understanding of the structure, function, and signaling mechanisms of the membrane bound GHR. 43 refs.

  14. EXTRACELLULAR HEAT SHOCK PROTEINS: A NEW LOCATION, A NEW FUNCTION

    PubMed Central

    De Maio, Antonio; Vazquez, Daniel

    2015-01-01

    The expression of heat shock proteins (hsp) is a basic and well conserved cellular response to an array of stresses. These proteins are involved in the repair of cellular damage induced by the stress, which is necessary for the salutary resolution from the insult. Moreover, they confer protection from subsequent insults, which has been coined stress tolerance. Since these proteins are expressed in subcellular compartments, it was thought that their function during stress conditions was circumscribed to the intracellular environment. However, it is now well established that hsp can also be present outside cells where they appear to display a function different than the well understood chaperone role. Extracellular hsp act as alert stress signals priming other cells, particularly of the immune system, to avoid the propagation of the insult and favor resolution. Since the majority of hsp do not possess a secretory peptide signal, they are likely be exported by a non-classical secretory pathway. Different mechanisms have been proposed to explain the export of hsp, including translocation across the plasma membrane and release associated with lipid vesicles, as well as the passive release after cell death by necrosis. Extracellular hsp appear in various flavors, including membrane-bound and membrane-free forms. All of these variants of extracellular hsp suggest that their interactions with cells may be quite diverse, both in target cell types and the activation signaling pathways. This review addresses some of our current knowledge about the release and relevance of extracellular hsp. PMID:23807250

  15. β2 Adrenergic Receptor Fluorescent Protein Fusions Traffic to the Plasma Membrane and Retain Functionality

    PubMed Central

    Bubnell, Jaclyn; Pfister, Patrick; Sapar, Maria L.; Rogers, Matthew E.; Feinstein, Paul

    2013-01-01

    Green fluorescent protein (GFP) has proven useful for the study of protein interactions and dynamics for the last twenty years. A variety of new fluorescent proteins have been developed that expand the use of available excitation spectra. We have undertaken an analysis of seven of the most useful fluorescent proteins (XFPs), Cerulean (and mCerulean3), Teal, GFP, Venus, mCherry and TagRFP657, as fusions to the archetypal G-protein coupled receptor, the β2 adrenergic receptor (β2AR). We have characterized these β2AR::XFP fusions in respect to membrane trafficking and G-protein activation. We noticed that in the mouse neural cell line, OP 6, that membrane bound β2AR::XFP fusions robustly localized in the filopodia identical to gap::XFP fusions. All β2AR::XFP fusions show responses indistinguishable from each other and the non-fused form after isoprenaline exposure. Our results provide a platform by which G-protein coupled receptors can be dissected for their functionality. PMID:24086401

  16. Guanylylation-competent replication proteins of Tomato mosaic virus are disulfide-linked.

    PubMed

    Nishikiori, Masaki; Meshi, Tetsuo; Ishikawa, Masayuki

    2012-12-01

    The 130-kDa and 180-kDa replication proteins of Tomato mosaic virus (ToMV) covalently bind guanylate and transfer it to the 5' end of RNA to form a cap. We found that guanylylation-competent ToMV replication proteins are in membrane-bound, disulfide-linked complexes. Guanylylation-competent replication proteins of Brome mosaic virus and Cucumber mosaic virus behaved similarly. To investigate the roles of disulfide bonding in the functioning of ToMV replication proteins, each of the 19 cysteine residues in the 130-kDa protein was replaced by a serine residue. Interestingly, three mutant proteins (C179S, C186S and C581S) failed not only to be guanylylated, but also to bind to the replication template and membranes. These mutants could trans-complement viral RNA replication. Considering that ToMV replication proteins recognize the replication templates, bind membranes, and are guanylylated in the cytoplasm that provides a reducing condition, we discuss the roles of cysteine residues and disulfide bonds in ToMV RNA replication. PMID:23062762

  17. Structural Bioinformatics Inspection of neXtProt PE5 Proteins in the Human Proteome.

    PubMed

    Dong, Qiwen; Menon, Rajasree; Omenn, Gilbert S; Zhang, Yang

    2015-09-01

    One goal of the Human Proteome Project is to identify at least one protein product for each of the ∼20,000 human protein-coding genes. As of October 2014, however, there are 3564 genes (18%) that have no or insufficient evidence of protein existence (PE), as curated by neXtProt; these comprise 2647 PE2-4 missing proteins and 616 PE5 dubious protein entries. We conducted a systematic examination of the 616 PE5 protein entries using cutting-edge protein structure and function modeling methods. Compared to a random sample of high-confidence PE1 proteins, the putative PE5 proteins were found to be over-represented in the membrane and cell surface proteins and peptides fold families. Detailed functional analyses show that most PE5 proteins, if expressed, would belong to transporters and receptors localized in the plasma membrane compartment. The results suggest that experimental difficulty in identifying membrane-bound proteins and peptides could have precluded their detection in mass spectrometry and that special enrichment techniques with improved sensitivity for membrane proteins could be important for the characterization of the PE5 "dark matter" of the human proteome. Finally, we identify 66 high scoring PE5 protein entries and find that six of them were reported in recent mass spectrometry databases; an illustrative annotation of these six is provided. This work illustrates a new approach to examine the potential folding and function of the dubious proteins comprising PE5, which we will next apply to the far larger group of missing proteins comprising PE2-4. PMID:26193931

  18. Structure, Function and On-Off Switching of a Core Unit Contact between CheA Kinase and CheW Adaptor Protein in the Bacterial Chemosensory Array: A Disulfide Mapping and TAM-IDS Study

    PubMed Central

    Natale, Andrew M.; Duplantis, Jane L.; Piasta, Kene N.; Falke, Joseph J.

    2014-01-01

    The ultrasensitive, ultrastable bacterial chemosensory array of Escherichia coli and Salmonella typhimurium is representative of the large, conserved family of sensory arrays that control the cellular chemotaxis of motile bacteria and Archaea. The core framework of the membrane-bound array is a lattice assembled from three components: a transmembrane receptor, a cytoplasmic His kinase (CheA), and a cytoplasmic adaptor protein (CheW). Structural studies in the field have revealed the global architecture of the array and complexes between specific components, but much remains to be learned about the essential protein-protein interfaces that define array structure and transmit signals between components. This study has focused on the structure, function and on-off switching of a key contact between the kinase and adaptor proteins in the working, membrane-bound array. Specifically, the study addressed interface 1 in the putative kinase-adaptor ring where subdomain 1 of the kinase regulatory domain contacts subdomain 2 of the adaptor protein. Two independent approaches – disulfide mapping and site-directed Trp and Ala mutagenesis – were employed to (i) test the structural model of interface 1 and (ii) investigate its functional roles in both stable kinase incorporation and receptor-regulated kinase on-off switching. Studies were carried out in functional, membrane-bound arrays or in live cells. The findings reveal that crystal structures of binary and ternary complexes accurately depict the native interface in its kinase-activating on state. Furthermore, the findings indicate that at least part of the interface becomes less closely packed in its kinase-inhibiting off state. Together, the evidence shows the interface has a dual structural and signaling function that is crucial for stable kinase incorporation into the array, for kinase activation in the array on state, and likely for attractant-triggered kinase on-off switching. A model is presented that describes the

  19. Protein and Signaling Networks in Vertebrate Photoreceptor Cells

    PubMed Central

    Koch, Karl-Wilhelm; Dell’Orco, Daniele

    2015-01-01

    Vertebrate photoreceptor cells are exquisite light detectors operating under very dim and bright illumination. The photoexcitation and adaptation machinery in photoreceptor cells consists of protein complexes that can form highly ordered supramolecular structures and control the homeostasis and mutual dependence of the secondary messengers cyclic guanosine monophosphate (cGMP) and Ca2+. The visual pigment in rod photoreceptors, the G protein-coupled receptor rhodopsin is organized in tracks of dimers thereby providing a signaling platform for the dynamic scaffolding of the G protein transducin. Illuminated rhodopsin is turned off by phosphorylation catalyzed by rhodopsin kinase (GRK1) under control of Ca2+-recoverin. The GRK1 protein complex partly assembles in lipid raft structures, where shutting off rhodopsin seems to be more effective. Re-synthesis of cGMP is another crucial step in the recovery of the photoresponse after illumination. It is catalyzed by membrane bound sensory guanylate cyclases (GCs) and is regulated by specific neuronal Ca2+-sensor proteins called guanylate cyclase-activating proteins (GCAPs). At least one GC (ROS-GC1) was shown to be part of a multiprotein complex having strong interactions with the cytoskeleton and being controlled in a multimodal Ca2+-dependent fashion. The final target of the cGMP signaling cascade is a cyclic nucleotide-gated (CNG) channel that is a hetero-oligomeric protein located in the plasma membrane and interacting with accessory proteins in highly organized microdomains. We summarize results and interpretations of findings related to the inhomogeneous organization of signaling units in photoreceptor outer segments. PMID:26635520

  20. Towards a Semen Proteome of the Dengue Vector Mosquito: Protein Identification and Potential Functions

    PubMed Central

    Sirot, Laura K.; Ribeiro, José M. C.; Kimura, Mari; Deewatthanawong, Prasit; Wolfner, Mariana F.; Harrington, Laura C.

    2011-01-01

    Background No commercially licensed vaccine or treatment is available for dengue fever, a potentially lethal infection that impacts millions of lives annually. New tools that target mosquito control may reduce vector populations and break the cycle of dengue transmission. Male mosquito seminal fluid proteins (Sfps) are one such target since these proteins, in aggregate, modulate the reproduction and feeding patterns of the dengue vector, Aedes aegypti. As an initial step in identifying new targets for dengue vector control, we sought to identify the suite of proteins that comprise the Ae. aegypti ejaculate and determine which are transferred to females during mating. Methodology and Principal Findings Using a stable-isotope labeling method coupled with proteomics to distinguish male- and female-derived proteins, we identified Sfps and sperm proteins transferred from males to females. Sfps were distinguished from sperm proteins by comparing the transferred proteins to sperm-enriched samples derived from testes and seminal vesicles. We identified 93 male-derived Sfps and 52 predicted sperm proteins that are transferred to females during mating. The Sfp protein classes we detected suggest roles in protein activation/inactivation, sperm utilization, and ecdysteroidogenesis. We also discovered that several predicted membrane-bound and intracellular proteins are transferred to females in the seminal fluids, supporting the hypothesis that Ae. aegypti Sfps are released from the accessory gland cells through apocrine secretion, as occurs in mammals. Many of the Ae. aegypti predicted sperm proteins were homologous to Drosophila melanogaster sperm proteins, suggesting conservation of their sperm-related function across Diptera. Conclusion and Significance This is the first study to directly identify Sfps transferred from male Ae. aegypti to females. Our data lay the groundwork for future functional analyses to identify individual seminal proteins that may trigger female post

  1. A Strongly Absorbing Class of Non-Natural Labels for Probing Protein Electrostatics and Solvation with FTIR and 2D IR Spectroscopies

    PubMed Central

    Woys, Ann Marie; Mukherjee, Sudipta S.; Skoff, David R.; Moran, Sean D.; Zanni, Martin T.

    2013-01-01

    A series of non-natural infrared probes is reported that consist of a metal-tricarbonyl modified with a -(CH2)n- linker and cysteine-specific leaving group. They can be site-specifically attached to proteins using mutagenesis and similar protocols for EPR spin labels, which have the same leaving group. We characterize the label’s frequencies and lifetimes using 2D IR spectroscopy in solvents of varying dielectric. The frequency range spans 10 cm−1, and the variation in lifetimes ranges from 6 to 19 ps, indicating that these probes are very sensitive to their environments. Also, we attached probes with -(CH2)-, -(CH2)3-, -(CH2)4- linkers to ubiquitin at positions 6 and 63 and collected spectra in aqueous buffer. The frequencies and lifetimes were correlated for 3C and 4C linkers, as they were in the solvents, but did not correlate for the 1C linker. We concluded that lifetime measures solvation, whereas frequency reflects the electrostatics of the environment, which in the case of the 1C linker is a measure of the protein electrostatic field. We also labeled V71C α-synuclein in buffer and membrane-bound. Unlike most other infrared labels, this label has extremely-strong cross-sections and so can be measured with 2D IR spectroscopy at sub-millimolar concentrations. We expect that these labels will find use in studying the structure and dynamics of membrane-bound, aggregated, and kinetically-evolving proteins for which high signal-to-noise at low protein concentrations is imperative. PMID:23537223

  2. Binding of the Three-Repeat Domain of Tau to Phospholipid Membranes induces an Aggregated-Like State of the Protein

    PubMed Central

    Künze, Georg; Barré, Patrick; Scheidt, Holger A.; Thomas, Lars; Eliezer, David; Huster, Daniel

    2013-01-01

    In patients with Alzheimer’s disease, the microtubule-associated protein tau is found aggregated into paired helical filaments (PHFs) in neurofibrillary deposits. In solution, tau is intrinsically unstructured. However, the tubulin binding domain consisting of three or four 31–32 amino acid repeat regions exhibits both helical and β-structure propensity and makes up the proteolysis resistant core of PHFs. Here, we studied the structure and dynamics of the three-repeat domain of tau (i.e. K19) when bound to membranes consisting of a phosphatidylcholine and phosphatidylserine mixture or phosphatidylserine alone. Tau K19 binds to phospholipid vesicles with submicromolar affinity as measured by fluorescence spectroscopy. The interaction is driven by electrostatic forces between the positively charged protein and the phospholipid head groups. The structure of the membrane-bound state of K19 was studied using CD spectroscopy and solid-state magic-angle spinning NMR spectroscopy. To this end, the protein was selectively 13C-labeled at all valine and leucine residues. Isotropic chemical shift values of tau K19 were consistent with a β-structure. In addition, motionally averaged 1H-13C dipolar couplings indicated a high rigidity of the protein backbone. The structure formation of K19 was also shown to depend on the charge density of the membrane. Phosphatidylserine membranes induced a gain in the α-helix structure along with an immersion of K19 into the phospholipid bilayer as indicated by a reduction of the lipid chain 2H NMR order parameter. Our results provide structural insights into the membrane-bound state of tau K19 and support a potential role of phospholipid membranes in mediating the physiological and pathological functions of tau. PMID:22521809

  3. Synchrotron X-ray footprinting as a method to visualize water in proteins.

    PubMed

    Gupta, Sayan; Feng, Jun; Chan, Leanne Jade G; Petzold, Christopher J; Ralston, Corie Y

    2016-09-01

    The vast majority of biomolecular processes are controlled or facilitated by water interactions. In enzymes, regulatory proteins, membrane-bound receptors and ion-channels, water bound to functionally important residues creates hydrogen-bonding networks that underlie the mechanism of action of the macromolecule. High-resolution X-ray structures are often difficult to obtain with many of these classes of proteins because sample conditions, such as the necessity of detergents, often impede crystallization. Other biophysical techniques such as neutron scattering, nuclear magnetic resonance and Fourier transform infrared spectroscopy are useful for studying internal water, though each has its own advantages and drawbacks, and often a hybrid approach is required to address important biological problems associated with protein-water interactions. One major area requiring more investigation is the study of bound water molecules which reside in cavities and channels and which are often involved in both the structural and functional aspects of receptor, transporter and ion channel proteins. In recent years, significant progress has been made in synchrotron-based radiolytic labeling and mass spectroscopy techniques for both the identification of bound waters and for characterizing the role of water in protein conformational changes at a high degree of spatial and temporal resolution. Here the latest developments and future capabilities of this method for investigating water-protein interactions and its synergy with other synchrotron-based methods are discussed. PMID:27577756

  4. Botrytis cinerea Protein O-Mannosyltransferases Play Critical Roles in Morphogenesis, Growth, and Virulence

    PubMed Central

    González, Mario; Brito, Nélida; Frías, Marcos; González, Celedonio

    2013-01-01

    Protein O-glycosylation is crucial in determining the structure and function of numerous secreted and membrane-bound proteins. In fungi, this process begins with the addition of a mannose residue by protein O-mannosyltransferases (PMTs) in the lumen side of the ER membrane. We have generated mutants of the three Botrytis cinerea pmt genes to study their role in the virulence of this wide-range plant pathogen. B. cinerea PMTs, especially PMT2, are critical for the stability of the cell wall and are necessary for sporulation and for the generation of the extracellular matrix. PMTs are also individually required for full virulence in a variety of hosts, with a special role in the penetration of intact plant leaves. The most significant case is that of grapevine leaves, whose penetration requires the three functional PMTs. Furthermore, PMT2 also contributes significantly to fungal adherence on grapevine and tobacco leaves. Analysis of extracellular and membrane proteins showed significant changes in the pattern of protein secretion and glycosylation by the pmt mutants, and allowed the identification of new protein substrates putatively glycosylated by specific PMTs. Since plants do no possess these enzymes, PMTs constitute a promising target in the development of novel control strategies against B. cinerea. PMID:23762450

  5. Selective enrichment of metal-binding proteins based on magnetic core/shell microspheres functionalized with metal cations.

    PubMed

    Fang, Caiyun; Zhang, Lei; Zhang, Xiaoqin; Lu, Haojie

    2015-06-21

    Metal binding proteins play many important roles in a broad range of biological processes. Characterization of metal binding proteins is important for understanding their structure and biological functions, thus leading to a clear understanding of metal associated diseases. The present study is the first to investigate the effectiveness of magnetic microspheres functionalized with metal cations (Ca(2+), Cu(2+), Zn(2+) and Fe(3+)) as the absorbent matrix in IMAC technology to enrich metal containing/binding proteins. The putative metal binding proteins in rat liver were then globally characterized by using this strategy which is very easy to handle and can capture a number of metal binding proteins effectively. In total, 185 putative metal binding proteins were identified from rat liver including some known less abundant and membrane-bound metal binding proteins such as Plcg1, Acsl5, etc. The identified proteins are involved in many important processes including binding, catalytic activity, translation elongation factor activity, electron carrier activity, and so on. PMID:25913209

  6. Structural studies of human glioma pathogenesis-related protein 1

    PubMed Central

    Asojo, Oluwatoyin A.; Koski, Raymond A.; Bonafé, Nathalie

    2011-01-01

    Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but is barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide that directs its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins) domain and a transmembrane domain. GLIPR1 is currently being investigated as a candidate for prostate cancer gene therapy and for glioblastoma targeted therapy. Crystal structures of a truncated soluble domain of the human GLIPR1 protein (sGLIPR1) solved by molecular replacement using a truncated polyalanine search model of the CAP domain of stecrisp, a snake-venom cysteine-rich secretory protein (CRISP), are presented. The correct molecular-replacement solution could only be obtained by removing all loops from the search model. The native structure was refined to 1.85 Å resolution and that of a Zn2+ complex was refined to 2.2 Å resolution. The latter structure revealed that the putative binding cavity coordinates Zn2+ similarly to snake-venom CRISPs, which are involved in Zn2+-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop/turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. A model is also proposed for the structure of full-length membrane-bound GLIPR1. PMID:21931216

  7. Rhamnose Links Moonlighting Proteins to Membrane Phospholipid in Mycoplasmas.

    PubMed

    Daubenspeck, James M; Liu, Runhua; Dybvig, Kevin

    2016-01-01

    Many proteins that have a primary function as a cytoplasmic protein are known to have the ability to moonlight on the surface of nearly all organisms. An example is the glycolytic enzyme enolase, which can be found on the surface of many types of cells from bacteria to human. Surface enolase is not enzymatic because it is monomeric and oligomerization is required for glycolytic activity. It can bind various molecules and activate plasminogen. Enolase lacks a signal peptide and the mechanism by which it attaches to the surface is unknown. We found that treatment of whole cells of the murine pathogen Mycoplasma pulmonis with phospholipase D released enolase and other common moonlighting proteins. Glycostaining suggested that the released proteins were glycosylated. Cytoplasmic and membrane-bound enolase was isolated by immunoprecipitation. No post-translational modification was detected on cytoplasmic enolase, but membrane enolase was associated with lipid, phosphate and rhamnose. Treatment with phospholipase released the lipid and phosphate from enolase but not the rhamnose. The site of rhamnosylation was identified as a glutamine residue near the C-terminus of the protein. Rhamnose has been found in all species of mycoplasma examined but its function was previously unknown. Mycoplasmas are small bacteria with have no peptidoglycan, and rhamnose in these organisms is also not associated with polysaccharide. We suggest that rhamnose has a central role in anchoring proteins to the membrane by linkage to phospholipid, which may be a general mechanism for the membrane association of moonlighting proteins in mycoplasmas and perhaps other bacteria. PMID:27603308

  8. Redistribution of the discs large tumor suppressor protein during mitosis.

    PubMed

    Massimi, Paola; Gardiol, Daniela; Roberts, Sally; Banks, Lawrence

    2003-11-01

    Drosophila discs large (Dlg) has been shown to be an essential regulator of cell polarity and attachment, and is classified as a potential tumour suppressor in higher eukaryotes. Human Dlg is expressed in epithelial cells at sites of cell-cell contact and acts as a negative regulator of cell growth. Although hDlg has been shown to be phosphorylated during mitosis, little is known about its activity during this stage of the cell cycle. To investigate this further we have analysed in detail the pattern of hDlg expression during mitotic cell division. In early mitosis there is a marked increase in membrane-bound hDlg which is then retained throughout mitosis, while during cytokinesis, there is a specific concentration of hDlg at the midbody. Using mutants of Dlg we show that this is mediated by sequences in the carboxy terminal region of Dlg, but it does not require the SH3 or PDZ domains, and is independent of binding to protein 4.1. Finally, using a mutant of Dlg that consists of just this carboxy terminal region of the protein, we show that it can compete with endogenous hDlg for midbody accumulation, and this mutant also gives rise to altered cell growth. We conclude that localisation of Dlg to the midbody indicates a role for Dlg at this critical point in cytokinesis. PMID:14567986

  9. The statolith compartment in Chara rhizoids contains carbohydrate and protein

    NASA Technical Reports Server (NTRS)

    Wang-Cahill, F.; Kiss, J. Z.

    1995-01-01

    In contrast to higher plants, the alga Chara has rhizoids with single membrane-bound compartments that function as statoliths in gravity perception. Previous work has demonstrated that these statoliths contain barium sulfate crystals. In this study, we show that statoliths in Chara rhizoids react with a Coomassie Brilliant Blue cytochemical stain for proteins. While statoliths did not react with silver methenamine carbohydrate cytochemistry, the monoclonal antibody CCRC-M2, which is against a carbohydrate (sycamore-maple rhamnogalacturonan I), labeled the statolith compartment. These results demonstrate that in addition to barium sulfate, statoliths in Chara rhizoids have an organic matrix that consists of protein and carbohydrate moieties. Since the statoliths were silver methenamine negative, the carbohydrate in this compartment could be a 3-linked polysaccharide. CCRC-M2 also labeled Golgi cisternae, Golgi-associated vesicles, apical vesicles, and cell walls in the rhizoids. The specificity of CCRC-M2 immunolabeling was verified by several control experiments, including the demonstration that labeling was abolished when the antibody was preabsorbed with its antigen. Since in this and a previous study (John Z. Kiss and L. Andrew Staehelin, American Journal of Botany 80: 273-282, 1993) antibodies against higher plant carbohydrates crossreacted with cell walls of Chara in a specific manner, Characean algae may be a useful model system in biochemical and molecular studies of cell walls.

  10. The cellular prion protein and its role in Alzheimer disease

    PubMed Central

    Irujo, A; Cuadrado-Tejedor, M; Paternain, B; Moleres, FJ; Ferrer, V

    2009-01-01

    The cellular prion protein (PrPC) is a membrane-bound glycoprotein especially abundant in the central nervous system (CNS). The scrapie prion protein (PrPSc, also termed prions) is responsible of transmissible spongiform encephalopathies (TSE), a group of neurodegenerative diseases which affect humans and other mammal species, although the presence of PrPC is needed for the establishment and further evolution of prions. The present work compares the expression and localization of PrPC between healthy human brains and those suffering from Alzheimer disease (AD). In both situations we have observed a rostrocaudal decrease in the amount of PrPC within the CNS, both by immunoblotting and immunohistochemistry techniques. PrPC is higher expressed in our control brains than in AD cases. There was a neuronal loss and astogliosis in our AD cases. There was a tendency of a lesser expression of PrPC in AD cases than in healthy ones. And in AD cases, the intensity of the expression of the unglycosylated band is higher than the di- and monoglycosylated bands. With regards to amyloid plaques, those present in AD cases were positively labeled for PrPC, a result which is further supported by the presence of PrPC in the amyloid plaques of a transgenic line of mice mimicking AD. The work was done according to Helsinki Declaration of 1975, and approved by the Ethics Committee of the Faculty of Medicine of the University of Navarre. PMID:19556894

  11. Protein import, replication, and inheritance of a vestigial mitochondrion.

    PubMed

    Regoes, Attila; Zourmpanou, Danai; León-Avila, Gloria; van der Giezen, Mark; Tovar, Jorge; Hehl, Adrian B

    2005-08-26

    Mitochondrial remnant organelles (mitosomes) that exist in a range of "amitochondrial" eukaryotic organisms represent ideal models for the study of mitochondrial evolution and for the establishment of the minimal set of proteins required for the biogenesis of an endosymbiosis-derived organelle. Giardia intestinalis, often described as the earliest branching eukaryote, contains double membrane-bounded structures involved in iron-sulfur cluster biosynthesis, an essential function of mitochondria. Here we present evidence that Giardia mitosomes also harbor Cpn60, mtHsp70, and ferredoxin and that despite their advanced state of reductive evolution they have retained vestiges of presequence-dependent and -independent protein import pathways akin to those that operate in mammalian mitochondria. Although import of IscU and ferredoxin is still reliant on their amino-terminal presequences, targeting of Giardia Cpn60, IscS, or mtHsp70 into mitosomes no longer requires cleavable presequences, a derived feature from their mitochondrial homologues. In addition, we found that division and segregation of a single centrally positioned mitosome tightly associated with the microtubular cytoskeleton is coordinated with the cell cycle, whereas peripherally located mitosomes are inherited into daughter cells stochastically. PMID:15985435

  12. Interfacial assembly of protein-polymer nano-conjugates into stimulus-responsive biomimetic protocells.

    PubMed

    Huang, Xin; Li, Mei; Green, David C; Williams, David S; Patil, Avinash J; Mann, Stephen

    2013-01-01

    The mechanism of spontaneous assembly of microscale compartments is a central question for the origin of life, and has technological repercussions in diverse areas such as materials science, catalysis, biotechnology and biomedicine. Such compartments need to be semi-permeable, structurally robust and capable of housing assemblages of functional components for internalized chemical transformations. In principle, proteins should be ideal building blocks for the construction of membrane-bound compartments but protein vesicles with cell-like properties are extremely rare. Here we present an approach to the interfacial assembly of protein-based micro-compartments (proteinosomes) that are delineated by a semi-permeable, stimulus-responsive, enzymatically active, elastic membrane consisting of a closely packed monolayer of conjugated protein-polymer building blocks. The proteinosomes can be dispersed in oil or water, thermally cycled to temperatures of 70 °C, and partially dried and re-inflated without loss of structural integrity. As a consequence, they exhibit protocellular properties such as guest molecule encapsulation, selective permeability, gene-directed protein synthesis and membrane-gated internalized enzyme catalysis. PMID:23896993

  13. Wherever I may roam: protein and membrane trafficking in P. falciparum-infected red blood cells.

    PubMed

    Deponte, Marcel; Hoppe, Heinrich C; Lee, Marcus C S; Maier, Alexander G; Richard, Dave; Rug, Melanie; Spielmann, Tobias; Przyborski, Jude M

    2012-12-01

    Quite aside from its immense importance as a human pathogen, studies in recent years have brought to light the fact that the malaria parasite Plasmodium falciparum is an interesting eukaryotic model system to study protein trafficking. Studying parasite cell biology often reveals an overrepresentation of atypical cell biological features, possibly driven by the parasites' need to survive in an unusual biological niche. Malaria parasites possess uncommon cellular compartments to which protein traffic must be directed, including secretory organelles such as rhoptries and micronemes, a lysosome-like compartment referred to as the digestive vacuole and a complex (four membrane-bound) plastid, the apicoplast. In addition, the parasite must provide proteins to extracellular compartments and structures including the parasitophorous vacuole, the parasitophorous vacuolar membrane, the Maurer's clefts and both cytosol and plasma membrane of the host cell, the mature human red blood cell. Although some of these unusual destinations are possessed by other cell types, only Plasmodium parasites contain them all within one cell. Here we review what is known about protein and membrane transport in the P. falciparum-infected cell, highlighting novel features of these processes. A growing body of evidence suggests that this parasite is a real "box of tricks" with regards to protein traffic. Possibly, these tricks may be turned against the parasite by exploiting them as novel therapeutic targets. PMID:23043991

  14. Proteomics profiling of cholangiocarcinoma exosomes: A potential role of oncogenic protein transferring in cancer progression.

    PubMed

    Dutta, Suman; Reamtong, Onrapak; Panvongsa, Wittaya; Kitdumrongthum, Sarunya; Janpipatkul, Keatdamrong; Sangvanich, Polkit; Piyachaturawat, Pawinee; Chairoungdua, Arthit

    2015-09-01

    Cholangiocarcinoma (CCA), a common primary malignant tumor of bile duct epithelia, is highly prevalent in Asian countries and unresponsive to chemotherapeutic drugs. Thus, a newly recognized biological entity for early diagnosis and treatment is highly needed. Exosomes are small membrane bound vesicles found in body fluids and released by most cell types including cancer cells. The vesicles contain specific subset of proteins and nucleic acids corresponding to cell types and play essential roles in pathophysiological processes. The present study aimed to assess the protein profiles of CCA-derived exosomes and their potential roles. We have isolated exosomes from CCA cells namely KKU-M213 and KKU-100 derived from Thai patients and their roles were investigated by incubation with normal human cholangiocyte (H69) cells. Exosomes were internalized into H69 cells and had no effects on viability or proliferation of the host cells. Interestingly, the exosomes from KKU-M213 cells only induced migration and invasion of H69 cells. Proteomic analysis of the exosomes from KKU-M213 cells disclosed multiple cancer related proteins that are not present in H69 exosomes. Consistent with the protein profile, treatment with KKU-M213 exosomes induced β-catenin and reduced E-cadherin expressions in H69 cells. Collectively, our results suggest that a direct cell-to-cell transfer of oncogenic proteins via exosomal pathway may be a novel mechanism for CCA progression and metastasis. PMID:26148937

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

  16. Structural Similarities between Thiamin-Binding Protein and Thiaminase-I Suggest a Common Ancestor

    SciTech Connect

    Soriano, Erika V.; Rajashankar, Kanagalaghatta R.; Hanes, Jeremiah W.; Bale, Shridhar; Begley, Tadhg P.; Ealick, Steven E.

    2008-06-30

    ATP-binding cassette (ABC) transporters are responsible for the transport of a wide variety of water-soluble molecules and ions into prokaryotic cells. In Gram-negative bacteria, periplasmic-binding proteins deliver ions or molecules such as thiamin to the membrane-bound ABC transporter. The gene for the thiamin-binding protein tbpA has been identified in both Escherichia coli and Salmonella typhimurium. Here we report the crystal structure of TbpA from E. coli with bound thiamin monophosphate. The structure was determined at 2.25 {angstrom} resolution using single-wavelength anomalous diffraction experiments, despite the presence of nonmerohedral twinning. The crystal structure shows that TbpA belongs to the group II periplasmic-binding protein family. Equilibrium binding measurements showed similar dissociation constants for thiamin, thiamin monophosphate, and thiamin pyrophosphate. Analysis of the binding site by molecular modeling demonstrated how TbpA binds all three forms of thiamin. A comparison of TbpA and thiaminase-I, a thiamin-degrading enzyme, revealed structural similarity between the two proteins, especially in domain 1, suggesting that the two proteins evolved from a common ancestor.

  17. Tight junction proteins in gallbladder epithelium: different expression in acute acalculous and calculous cholecystitis.

    PubMed

    Laurila, Jouko J; Karttunen, Tuomo; Koivukangas, Vesa; Laurila, Päivi A; Syrjälä, Hannu; Saarnio, Juha; Soini, Ylermi; Ala-Kokko, Tero I

    2007-06-01

    There is a paucity of information of tight junction (TJ) proteins in gallbladder epithelium, and disturbances in the structure of these proteins may play a role in the pathogenesis of acute acalculous cholecystitis (AAC) and acute calculous cholecystitis (ACC). Using immunohistochemistry, we investigated the expression of TJ proteins claudin-1, -2, -3, and -4, occludin, zonula occludens (ZO-1), and E-cadherin in 9 normal gallbladders, 30 gallbladders with AAC, and 21 gallbladders with ACC. The number of positive epithelial and endothelial cells and the intensity of the immunoreaction were determined. Membrane-bound and cytoplasmic immunoreactivities were separately assessed. We found that TJ proteins were uniformly expressed in normal gallbladder epithelium, with the exception of claudin-2, which was present in less than half of the cells. In AAC, expression of cytoplasmic occludin and claudin-1 were decreased, as compared with normal gallbladder. In ACC, expression of claudin-2 was increased, and expression of claudin-1, -3, and -4, occludin, and ZO-1 were decreased, as compared with normal gallbladder or AAC. We conclude that there are significant differences in expression of TJ proteins in AAC and ACC, supporting the idea that AAC represents a manifestation of systemic inflammatory disease, whereas ACC is a local inflammatory and often infectious disease. PMID:17283368

  18. Binding Affinity Effects on Physical Characteristics of a Model Phase-Separated Protein Droplet

    NASA Astrophysics Data System (ADS)

    Chuang, Sara; Banani, Salman; Rosen, Michael; Brangwynne, Clifford

    2015-03-01

    Non-membrane bound organelles are associated with a range of biological functions. Several of these structures exhibit liquid-like properties, and may represent droplets of phase-separated RNA and/or proteins. These structures are often enriched in multi-valent molecules, however little is known about the interactions driving the assembly, properties, and function. Here, we address this question using a model multi-valent protein system consisting of repeats of Small Ubiquitin-like Modifier (SUMO) protein and a SUMO-interacting motif (SIM). These proteins undergo phase separation into liquid-like droplets. We combine microrheology and quantitative microscopy to determine affect of binding affinity on the viscosity, density and surface tension of these droplets. We also use fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and partitioning experiments to probe the structure and dynamics within these droplets. Our results shed light on how inter-molecular interactions manifests in droplet properties, and lay the groundwork for a comprehensive biophysical picture of intracellular RNA/protein organelles.

  19. The role of lactoferrin binding protein B in mediating protection against human lactoferricin.

    PubMed

    Morgenthau, Ari; Livingstone, Margaret; Adamiak, Paul; Schryvers, Anthony B

    2012-06-01

    Bacteria that inhabit the mucosal surfaces of the respiratory and genitourinary tracts of mammals encounter an iron-deficient environment because of iron sequestration by the host iron-binding proteins transferrin and lactoferrin. Lactoferrin is also present in high concentrations at sites of inflammation where the cationic, antimicrobial peptide lactoferricin is produced by proteolysis of lactoferrin. Several Gram-negative pathogens express a lactoferrin receptor that enables the bacteria to use lactoferrin as an iron source. The receptor is composed of an integral membrane protein, lactoferrin binding protein A (LbpA), and a membrane-bound lipoprotein, lactoferrin binding protein B (LbpB). LbpA is essential for growth with lactoferrin as the sole iron source, whereas the role of LbpB in iron acquisition is not yet known. In this study, we demonstrate that LbpB from 2 different species is capable of providing protection against the killing activity of a human lactoferrin-derived peptide. We investigated the prevalence of lactoferrin receptors in bacteria and examined their sequence diversity. We propose that the protection against the cationic antimicrobial human lactoferrin-derived peptide is associated with clusters of negatively charged amino acids in the C-terminal lobe of LbpB that is a common feature of this protein. PMID:22332888

  20. Assembly, Properties and Function of Synthetic Phase-Separated RNA/Protein Organelles

    NASA Astrophysics Data System (ADS)

    Taylor, Nicole; Elbaum, Shana; Stone, Howard; Brangwynne, Clifford

    2015-03-01

    Non-membrane bound RNA/protein (RNP) bodies play a key role in cellular RNA processing steps. Many RNA helicases, required for RNA processing, are key components of RNPs. Consistent with this, a purified RNA helicase, Laf-1, exhibits a salt and protein concentration dependent phase separation in vitro, resulting in liquid-like droplets. We use such synthetic RNPs to study the biophysics of RNP assembly, and to elucidate the link between their physical properties and function. To accomplish this, we are developing custom microfluidic devices to measure biophysical properties, nucleation and growth kinetics, and RNA processing function of droplets. We measure droplet viscosity by applying a shear stress to protein droplets that adhere to the channel wall; measurements are consistent with those taken using a particle microrheology approach. We also monitor and control protein droplet nucleation using oil/water emulsions. Our results provide a new platform for addressing how the cell regulates organelle assembly and properties through protein, RNA, and ATP concentration. We anticipate that these findings will offer insight into the contribution of RNPs in key RNA processing functions in the cell.

  1. Protein targeting to the nuclear pore. What can we learn from plants. [Nuclear pore complex

    SciTech Connect

    Smith, H.M.S.; Raikhel, N.V. . DOE Plant Research Lab.)

    1999-04-01

    Characteristic of eukaryotic cells are the numerous types of membrane-bound organelles or compartments found in the cytoplasm, with each type carrying out an essential function for the cell. The spatial separation of proteins and biochemical pathways typical of the various types of organelles requires selective targeting apparatuses. Because each type of organelle contains its own targeting apparatus, proteins destined for a particular organelle must contain the proper targeting signal(s) for entry. These signal-dependent targeting pathways ensure that proteins are targeted to the proper organelle. Understanding how proteins are targeted to the different types of organelles is an important goal in the field of cell biology. In plants recent studies have highlighted a number of unusual features, and as the understanding of import in plants increases, the authors have gained new insights, such as a model for the targeting of proteins from the cytoplasm to the NPC. These advances will contribute to further expansion of the knowledge of nuclear import in eukaryotes.

  2. The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

    PubMed Central

    Brunati, Martina; Perucca, Simone; Han, Ling; Cattaneo, Angela; Consolato, Francesco; Andolfo, Annapaola; Schaeffer, Céline; Olinger, Eric; Peng, Jianhao; Santambrogio, Sara; Perrier, Romain; Li, Shuo; Bokhove, Marcel; Bachi, Angela; Hummler, Edith; Devuyst, Olivier; Wu, Qingyu; Jovine, Luca; Rampoldi, Luca

    2015-01-01

    Uromodulin is the most abundant protein in the urine. It is exclusively produced by renal epithelial cells and it plays key roles in kidney function and disease. Uromodulin mainly exerts its function as an extracellular matrix whose assembly depends on a conserved, specific proteolytic cleavage leading to conformational activation of a Zona Pellucida (ZP) polymerisation domain. Through a comprehensive approach, including extensive characterisation of uromodulin processing in cellular models and in specific knock-out mice, we demonstrate that the membrane-bound serine protease hepsin is the enzyme responsible for the physiological cleavage of uromodulin. Our findings define a key aspect of uromodulin biology and identify the first in vivo substrate of hepsin. The identification of hepsin as the first protease involved in the release of a ZP domain protein is likely relevant for other members of this protein family, including several extracellular proteins, as egg coat proteins and inner ear tectorins. DOI: http://dx.doi.org/10.7554/eLife.08887.001 PMID:26673890

  3. The HtrA/DegP family protease MamE is a bifunctional protein with roles in magnetosome protein localization and magnetite biomineralization

    PubMed Central

    Quinlan, Anna; Murat, Dorothée; Vali, Hojatollah; Komeili, Arash

    2011-01-01

    Summary Magnetotactic bacteria contain nanometer-sized, membrane-bound organelles, called magnetosomes, which are tasked with the biomineralization of small crystals of the iron oxide magnetite allowing the organism to use geomagnetic field lines for navigation. A key player in this process is the HtrA/DegP family protease MamE. In its absence, Magnetospirillum magneticum str AMB-1 is able to form magnetosome membranes but not magnetite crystals, a defect previously linked to the mislocalization of magnetosome proteins. In this work we use a directed genetic approach to find that MamE, and another predicted magnetosome-associated protease, MamO, likely function as proteases in vivo. However, as opposed to the complete loss of mamE where no biomineralization is observed, the protease-deficient variant of this protein still supports the initiation and formation of small, 20 nm-sized crystals of magnetite, too small to hold a permanent magnetic dipole moment. This analysis also reveals that MamE is a bifunctional protein with a protease-independent role in magnetosome protein localization and a protease-dependent role in maturation of small magnetite crystals. Together these results imply the existence of a previously unrecognized “checkpoint” in biomineralization where MamE moderates the completion of magnetite formation and thus committal to magneto-aerotaxis as the organism’s dominant mode of navigating the environment. PMID:21414040

  4. Nerve Growth Factor Increases mRNA Levels for the Prion Protein and the β -amyloid Protein Precursor in Developing Hamster Brain

    NASA Astrophysics Data System (ADS)

    Mobley, William C.; Neve, Rachael L.; Prusiner, Stanley B.; McKinley, Michael P.

    1988-12-01

    Deposition of amyloid filaments serves as a pathologic hallmark for some neurodegenerative disorders. The prion protein (PrP) is found in amyloid of animals with scrapie and humans with Creutzfeldt-Jakob disease; the β protein is present in amyloid deposits in Alzheimer disease and Down syndrome patients. These two proteins are derived from precursors that in the brain are expressed primarily in neurons and are membrane bound. We found that gene expression for PrP and the β -protein precursor (β -PP) is regulated in developing hamster brain. Specific brain regions showed distinct patterns of ontogenesis for PrP and β -PP mRNAs. The increases in PrP and β -PP mRNAs in developing basal forebrain coincided with an increase in choline acetyltransferase activity, raising the possibility that these markers might be coordinately controlled in cholinergic neurons and regulated by nerve growth factor (NGF). Injections of NGF into the brains of neonatal hamsters increased both PrP and β -PP mRNA levels. Increased PrP and β -PP mRNA levels induced by NGF were confined to regions that contain NGF-responsive cholinergic neurons and were accompanied by elevations in choline acetyltransferase. It remains to be established whether or not exogenous NGF acts to increase PrP and β -PP gene expression selectively in forebrain cholinergic neurons in the developing hamster and endogenous NGF regulates expression of these genes.

  5. Favism: effect of divicine on rat erythrocyte sulfhydryl status, hexose monophosphate shunt activity, morphology, and membrane skeletal proteins.

    PubMed

    McMillan, D C; Bolchoz, L J; Jollow, D J

    2001-08-01

    Favism is an acute anemic crisis that can occur in susceptible individuals who ingest fava beans. The fava bean pyrimidine aglycone divicine has been identified as a hemotoxic constituent; however, its mechanism of toxicity remains unknown. We have shown recently that divicine can induce a favic-like response in rats and that divicine is directly toxic to rat red cells. In the present study, we have examined the effect of hemotoxic concentrations of divicine on rat erythrocyte sulfhydryl status, hexose monophosphate (HMP) shunt activity, morphology, and membrane skeletal proteins. In vitro exposure of rat red cells to divicine markedly stimulated HMP shunt activity and resulted in depletion of reduced glutathione with concomitant formation of glutathione-protein mixed-disulfides. Examination of divicine-treated red cells by scanning electron microscopy revealed transformation of the cells to an extreme echinocytic morphology. SDS-PAGE and immunoblotting analysis of the membrane skeletal proteins indicated that hemotoxicity was associated with the apparent loss of skeletal protein bands 2.1, 3, and 4.2, and the appearance of membrane-bound hemoglobin. Treatment of divicine-damaged red cells with dithiothreitol reversed the protein changes, which indicated that the observed alterations were due primarily to the formation of disulfide-linked hemoglobin-skeletal protein adducts. The data suggest that oxidative modification of hemoglobin and membrane skeletal proteins by divicine may be key events in the mechanism underlying favism. PMID:11452148

  6. Deletion of potD, encoding a putative spermidine-binding protein, results in a complex phenotype in Legionella pneumophila.

    PubMed

    Nasrallah, Gheyath K; Abdelhady, Hany; Tompkins, Nicholas P; Carson, Kaitlyn R; Garduño, Rafael A

    2014-07-01

    L. pneumophila is an intracellular pathogen that replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV). We previously observed that the polyamine spermidine, produced by host cells or added exogenously, enhances the intracellular growth of L. pneumophila. To study this enhancing effect and determine whether polyamines are used as nutrients, we deleted potD from L. pneumophila strain JR32. The gene potD encodes a spermidine-binding protein that in other bacteria is essential for the function of the PotABCD polyamine transporter. Deletion of potD did not affect L. pneumophila growth in vitro in the presence or absence of spermidine and putrescine, suggesting that PotD plays a redundant or no role in polyamine uptake. However, deletion of potD resulted in a puzzlingly complex phenotype that included defects in L. pneumophila's ability to form filaments, tolerate Na(+), associate with macrophages and amoeba, recruit host vesicles to the LCV, and initiate intracellular growth. Moreover, the ΔpotD mutant was completely unable to grow in L929 cells treated with a pharmacological inhibitor of spermidine synthesis. These complex and disparate effects suggest that the L. pneumophila potD encodes either: (i) a multifunctional protein, (ii) a protein that interacts with, or regulates a, multifunctional protein, or (iii) a protein that contributes (directly or indirectly) to a regulatory network. Protein function studies with the L. pneumophila PotD protein are thus warranted. PMID:24928741

  7. Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region.

    PubMed Central

    Bienz, K; Egger, D; Troxler, M; Pasamontes, L

    1990-01-01

    Transcriptionally active replication complexes bound to smooth membrane vesicles were isolated from poliovirus-infected cells. In electron microscopic, negatively stained preparations, the replication complex appeared as an irregularly shaped, oblong structure attached to several virus-induced vesicles of a rosettelike arrangement. Electron microscopic immunocytochemistry of such preparations demonstrated that the poliovirus replication complex contains the proteins coded by the P2 genomic region (P2 proteins) in a membrane-associated form. In addition, the P2 proteins are also associated with viral RNA, and they can be cross-linked to viral RNA by UV irradiation. Guanidine hydrochloride prevented the P2 proteins from becoming membrane bound but did not change their association with viral RNA. The findings allow the conclusion that the protein 2C or 2C-containing precursor(s) is responsible for the attachment of the viral RNA to the vesicular membrane and for the spatial organization of the replication complex necessary for its proper functioning in viral transcription. A model for the structure of the viral replication complex and for the function of the 2C-containing P2 protein(s) and the vesicular membranes is proposed. Images PMID:2154600

  8. Golgi localized barley MTP8 proteins facilitate Mn transport.

    PubMed

    Pedas, Pai; Schiller Stokholm, Michaela; Hegelund, Josefine Nymark; Ladegård, Anne Hald; Schjoerring, Jan Kofod; Husted, Søren

    2014-01-01

    Many metabolic processes in plants are regulated by manganese (Mn) but limited information is available on the molecular mechanisms controlling cellular Mn homeostasis. In this study, a yeast assay was used to isolate and characterize two genes, MTP8.1 and MTP8.2, which encode membrane-bound proteins belonging to the cation diffusion facilitator (CDF) family in the cereal species barley (Hordeum vulgare). Transient expression in onion epidermal cells showed that MTP8.1 and MTP8.2 proteins fused to the green fluorescent protein (GFP) are localized to Golgi. When heterologously expressed in yeast, MTP8.1 and MTP8.2 were found to be Mn transporters catalysing Mn efflux in a similar manner as the Golgi localized endogenous yeast protein Pmr1p. The level of MTP8.1 transcripts in barley roots increased with external Mn supply ranging from deficiency to toxicity, while MTP8.2 transcripts decreased under the same conditions, indicating non-overlapping functions for the two genes. In barley leaves, the expression of both MTP8 genes declined in response to toxic Mn additions to the roots suggesting a role in ensuring proper delivery of Mn to Golgi. Based on the above we suggest that barley MTP8 proteins are involved in Mn loading to the Golgi apparatus and play a role in Mn homeostasis by delivering Mn to Mn-dependent enzymes and/or by facilitating Mn efflux via secretory vesicles. This study highlights the importance of MTP transporters in Mn homeostasis and is the first report of Golgi localized Mn2+ transport proteins in a monocot plant species. PMID:25486417

  9. Golgi Localized Barley MTP8 Proteins Facilitate Mn Transport

    PubMed Central

    Pedas, Pai; Schiller Stokholm, Michaela; Hegelund, Josefine Nymark; Ladegård, Anne Hald; Schjoerring, Jan Kofod; Husted, Søren

    2014-01-01

    Many metabolic processes in plants are regulated by manganese (Mn) but limited information is available on the molecular mechanisms controlling cellular Mn homeostasis. In this study, a yeast assay was used to isolate and characterize two genes, MTP8.1 and MTP8.2, which encode membrane-bound proteins belonging to the cation diffusion facilitator (CDF) family in the cereal species barley (Hordeum vulgare). Transient expression in onion epidermal cells showed that MTP8.1 and MTP8.2 proteins fused to the green fluorescent protein (GFP) are localized to Golgi. When heterologously expressed in yeast, MTP8.1 and MTP8.2 were found to be Mn transporters catalysing Mn efflux in a similar manner as the Golgi localized endogenous yeast protein Pmr1p. The level of MTP8.1 transcripts in barley roots increased with external Mn supply ranging from deficiency to toxicity, while MTP8.2 transcripts decreased under the same conditions, indicating non-overlapping functions for the two genes. In barley leaves, the expression of both MTP8 genes declined in response to toxic Mn additions to the roots suggesting a role in ensuring proper delivery of Mn to Golgi. Based on the above we suggest that barley MTP8 proteins are involved in Mn loading to the Golgi apparatus and play a role in Mn homeostasis by delivering Mn to Mn-dependent enzymes and/or by facilitating Mn efflux via secretory vesicles. This study highlights the importance of MTP transporters in Mn homeostasis and is the first report of Golgi localized Mn2+ transport proteins in a monocot plant species. PMID:25486417

  10. Identification and analysis of potential targets in Streptococcus sanguinis using computer aided protein data analysis

    PubMed Central

    Chowdhury, Md Rabiul Hossain; Bhuiyan, Md IqbalKaiser; Saha, Ayan; Mosleh, Ivan MHAI; Mondol, Sobuj; Ahmed, C M Sabbir

    2014-01-01

    Purpose Streptococcus sanguinis is a Gram-positive, facultative aerobic bacterium that is a member of the viridans streptococcus group. It is found in human mouths in dental plaque, which accounts for both dental cavities and bacterial endocarditis, and which entails a mortality rate of 25%. Although a range of remedial mediators have been found to control this organism, the effectiveness of agents such as penicillin, amoxicillin, trimethoprim–sulfamethoxazole, and erythromycin, was observed. The emphasis of this investigation was on finding substitute and efficient remedial approaches for the total destruction of this bacterium. Materials and methods In this computational study, various databases and online software were used to ascertain some specific targets of S. sanguinis. Particularly, the Kyoto Encyclopedia of Genes and Genomes databases were applied to determine human nonhomologous proteins, as well as the metabolic pathways involved with those proteins. Different software such as Phyre2, CastP, DoGSiteScorer, the Protein Function Predictor server, and STRING were utilized to evaluate the probable active drug binding site with its known function and protein–protein interaction. Results In this study, among 218 essential proteins of this pathogenic bacterium, 81 nonhomologous proteins were accrued, and 15 proteins that are unique in several metabolic pathways of S. sanguinis were isolated through metabolic pathway analysis. Furthermore, four essentially membrane-bound unique proteins that are involved in distinct metabolic pathways were revealed by this research. Active sites and druggable pockets of these selected proteins were investigated with bioinformatic techniques. In addition, this study also mentions the activity of those proteins, as well as their interactions with the other proteins. Conclusion Our findings helped to identify the type of protein to be considered as an efficient drug target. This study will pave the way for researchers to

  11. Sialorphin, a natural inhibitor of rat membrane-bound neutral endopeptidase that displays analgesic activity

    PubMed Central

    Rougeot, Catherine; Messaoudi, Michaël; Hermitte, Véronique; Rigault, Anne Gaëlle; Blisnick, Thierry; Dugave, Christophe; Desor, Didier; Rougeon, François

    2003-01-01

    Sialorphin is an exocrine and endocrine signaling mediator, which has been identified by a genomic approach. It is synthesized predominantly in the submandibular gland and prostate of adult rats in response to androgen steroids and is released locally and systemically in response to stress. We now demonstrate that the cell surface molecule to which sialorphin binds in vivo in the rat kidney is the membrane-anchored neutral endopeptidase (neprilysin; NEP, EC 3.4.24.11). NEP plays an important role in nervous and peripheral tissues, as it turns off several peptide-signaling events at the cell surface. We show that sialorphin prevents spinal and renal NEP from breaking down its two physiologically relevant substrates, substance P and Met-enkephalin in vitro. Sialorphin inhibited the breakdown of substance P with an IC50 of 0.4–1 μM and behaved as a competitive inhibitor. In vivo, i.v. sialorphin elicited potent antinociceptive responses in two behavioral rat models of injury-induced acute and tonic pain, the pin-pain test and formalin test. The analgesia induced by 100–200 μg/kg doses of sialorphin required the activation of μ- and δ-opioid receptors, consistent with the involvement of endogenous opioid receptors in enkephalinergic transmission. We conclude that sialorphin protects endogenous enkephalins released after nociceptive stimuli by inhibiting NEP in vivo. Sialorphin is a natural systemically active regulator of NEP activity. Furthermore, our study provides evidence that it is a physiological modulator of pain perception after injury and might be the progenitor of a new class of therapeutic molecules. PMID:12835417

  12. An amperometric cholesterol biosensor based on epoxy resin membrane bound cholesterol oxidase

    PubMed Central

    Pundir, C.S.; Narang, Jagriti; Chauhan, Nidhi; Sharma, Preety; Sharma, Renu

    2012-01-01

    Background & objectives: The use of epoxy resin membrane as a support for immobilization of enzyme has resulted into improved sensitivity and stability of biosensors for uric acid, ascorbic acid and polyphenols. The present work was aimed to prepare an improved amperometric biosensor for determination of serum cholesterol required in the diagnostics and management of certain pathological conditions. Methods: Epoxy resin membrane with immobilized cholesterol oxidase was mounted on the cleaned platinum (Pt) electrode with a parafilm to construct a working electrode. This working electrode along with Ag/AgCl as reference and Ag wire as an auxiliary electrode were connected through a three terminal electrometer to construct a cholesterol biosensor. Results: The sensor showed optimum response within 25 sec at pH 7.0 and 45°C. The linear working range of biosensor was 1.0 to 8.0 mM cholesterol. Km and Imax for cholesterol were 5.0 mM and 9.09 μA, respectively. The biosensor measured serum cholesterol. The minimum detection limit of the sensor was 1.0 mM. The mean analytical recoveries of added cholesterol in serum (2.84 and 4.13 mM) were 91.4±2.8 and 92.3±3.1 per cent (n=6), respectively. Within and between assay coefficient of variation (CV) were <2 and <4 per cent, respectively. Biosensor had a storage life of 6 months at 4°C. Interpretation & conclusions: The use of epoxy resin membrane as a support for immobilization of cholesterol oxidase has resulted into an improved amperometric cholesterol biosensor. The present biosensor had an advantage over the existing biosensors as it worked at comparatively lower potential. PMID:23168704

  13. Divalent Counterions Tether Membrane-Bound Carbohydrates To Promote the Cohesion of Auditory Hair Bundles

    PubMed Central

    LeBoeuf, Adria C.; Ó Maoiléidigh, D.; Hudspeth, A.J.

    2011-01-01

    The cell membranes in the hair bundle of an auditory hair cell confront a difficult task as the bundle oscillates in response to sound: for efficient mechanotransduction, all the component stereocilia of the hair bundle must move essentially in unison, shearing at their tips yet maintaining contact without membrane fusion. One mechanism by which this cohesion might occur is counterion-mediated attachment between glycan components of apposed stereociliary membranes. Using capillary electrophoresis, we showed that the stereociliary glycocalyx acts as a negatively charged polymer brush. We found by force-sensing photomicrometry that the stereocilia formed elastic connections with one another to various degrees depending on the surrounding ionic environment and the presence of N-linked sugars. Mg2+ was a more potent mediator of attachment than was Ca2+. The forces between stereocilia produced chaotic stick-slip behavior. These results indicate that counterion-mediated interactions in the glycocalyx contribute to the stereociliary coherence that is essential for hearing. PMID:21943412

  14. Acoustic propagation in two-dimensional waveguide for membrane bounded ducts

    NASA Astrophysics Data System (ADS)

    Nawaz, Rab; Afzal, Muhammad; Ayub, Muhammad

    2015-02-01

    This work aims to investigate the mode-matching (MM) and low frequency approximation (LFA) solutions of a two dimensional waveguide problem with flanged junction. The relative merits of each approach are compared for the scattering of fluid-coupled wave. The boundary value problem involving higher order derivatives at boundaries becomes a non-Sturm-Liouville problem where the use of standard orthogonality relation (OR) enables the MM solution. The derivation of LFA is made which proves to be surprisingly accurate for structure-borne mode incident. In order to validate the truncated model expansion the distribution of power in duct regions is discussed and Gibbs oscillations are incorporated by reconstruction of the normal velocity field using Lanczos filter.

  15. Resveratrol interferes with the aggregation of membrane-bound human-IAPP: a molecular dynamics study.

    PubMed

    Lolicato, Fabio; Raudino, Antonio; Milardi, Danilo; La Rosa, Carmelo

    2015-03-01

    Amyloid aggregation of islet amyloid polypeptide (IAPP) in pancreatic tissues is a typical feature of type 2 diabetes mellitus. Resveratrol, a natural product extensively studied for its wide range of biological effects, has been shown to inhibit IAPP aggregation. However, the mechanism by which resveratrol inhibits IAPP aggregation is still far from complete elucidation. Now, an increasing knowledge of the mechanism of amyloid toxicity shifts the target of research towards the development of compounds which can prevent amyloid-mediated membrane damage rather than merely inhibit fiber formation. In this study we used all atom molecular dynamics to investigate the interaction of resveratrol with full-length human IAPP in a negatively charged membrane environment. Our results show that the presence of resveratrol induces the formation of secondary structures (sheets and helices) by inserting in a hydrophobic pocket between the interaction surface of two IAPP molecules in aqueous solution. On the other hand, resveratrol significantly perturbs the interaction of IAPP with negatively charged membranes by anchoring specific hydrophobic regions (23FGA25 and 32VGS34) of the peptide and forming a stable 1:2 IAPP:resveratrol complex at the water/membrane interphase. PMID:25638571

  16. Measurement of membrane-bound human heme oxygenase-1 activity using a chemically defined assay system.

    PubMed

    Huber, Warren J; Marohnic, Christopher C; Peters, Michelle; Alam, Jawed; Reed, James R; Masters, Bettie Sue Siler; Backes, Wayne L

    2009-04-01

    Heme oxygenase (HO) catalyzes heme degradation in a reaction requiring NADPH-cytochrome P450 reductase (CPR). Although most studies with HO used a soluble 30-kDa form, lacking the C-terminal membrane-binding region, recent reports show that the catalytic behavior of this enzyme is very different if this domain is retained; the overall activity was elevated 5-fold, and the K(m) for CPR decreased approximately 50-fold. The goal of these studies was to accurately measure HO activity using a coupled assay containing purified biliverdin reductase (BVR). This allows measurement of bilirubin formation after incorporation of full-length CPR and heme oxygenase-1 (HO-1) into a membrane environment. When rat liver cytosol was used as the source of partially purified BVR, the reaction remained linear for 2 to 3 min; however, the reaction was only linear for 10 to 30 s when an equivalent amount of purified, human BVR (hBVR) was used. This lack of linearity was not observed with soluble HO-1. Optimal formation of bilirubin was achieved with concentrations of bovine serum albumin (0.25 mg/ml) and hBVR (0.025-0.05 microM), but neither supplement increased the time that the reaction remained linear. Various concentrations of superoxide dismutase had no effect on the reaction; however, when catalase was included, the reactions were linear for at least 4 to 5 min, even at high CPR levels. These results not only show that HO-1-generated hydrogen peroxide leads to a decrease in HO-1 activity but also provide for a chemically defined system to be used to examine the function of full-length HO-1 in a membrane environment. PMID:19131520

  17. Effects of lipid peroxidation on membrane-bound enzymes of the endoplasmic reticulum

    PubMed Central

    Wills, E. D.

    1971-01-01

    1. Induction of the formation of lipid peroxide in suspensions of liver microsomal preparations by incubation with ascorbate or NADPH, or by treatment with ionizing radiation, leads to a marked decrease of the activity of glucose 6-phosphatase. 2. The effect of peroxidation can be imitated by treating microsomal suspensions with detergents such as deoxycholate or with phospholipases. 3. The substrate, glucose 6-phosphate, protects the glucose 6-phosphatase activity of microsomal preparations against peroxidation or detergents. 4. The loss of glucose 6-phosphatase activity is not due to the formation of hydroperoxide or formation of malonaldehyde or other breakdown products of peroxidation, all of which are not toxic to the enzyme. 5. All experiments lead to the conclusion that the loss of activity of glucose 6-phosphatase resulting from peroxidation is a consequence of loss of membrane structure essential for the activity of the enzyme. 6. In addition to glucose 6-phosphatase, oxidative demethylation of aminopyrine or p-chloro-N-methylaniline, hydroxylation of aniline, NADPH oxidation and menadione-dependent NADPH oxidation are also strongly inhibited by peroxidation. However, another group of enzymes separated with the microsomal fraction, including NAD+/NADP+ glycohydrolase, adenosine triphosphatase, esterase and NADH–cytochrome c reductase are not inactivated by peroxidation. This group is not readily inactivated by treatment with detergents. 7. Lipid peroxidation, by controlling membrane integrity, may exert a regulating effect on the oxidative metabolism and carbohydrate metabolism of the endoplasmic reticulum in vivo. PMID:4399403

  18. Photochemical energy conversion by membrane-bound photoredox systems. Final report

    SciTech Connect

    Tollin, G.

    1994-06-16

    This project has received DOE support since July 1, 1978. During this period, 40 papers have been published dealing predominantly with chlorophyll-photosensitized electron transfer reactions in a variety of media (solutions, polymer films, lipid bilayer membranes). The overall theme of this work has been to develop mechanistic strategies f or photochemical energy storage via chlorophyll, using the green plant photosynthetic system as a paradigm for designing in vitro systems. Microheterogeneous lipid vesicle suspensions allow ready application of time-resolved optical spectroscopy to follow the course of light-induced electron transfer processes. Both the yields and the lifetimes of electron transfer products were markedly improved in the vesicle systems. In subsequent studies, this compartmentalization was favorably manipulated by controlling the electrical charge on the membrane surface, by controlling the solubility properties of the acceptors, by varying the lipid composition, by using mediators to create a concentration gradient to carry electrons from within the bilayer to the aqueous medium, and by incorporating secondary electron acceptors into the aqueous phase.

  19. Postmortem changes in rat brain: studies on membrane-bound enzymes and receptors.

    PubMed

    Syapin, P J; Ritchie, T; Noble, L; Noble, E P

    1987-04-01

    The relationship between the stability of potential neurochemical markers and autolysis time was studied at 4 degrees C and 25 degrees C using postmortem brain samples from two rat strains. In general, qualitatively similar results were obtained with either N/Nih or Sprague-Dawley rats; however, quantitative differences were often observed, particularly in regard to benzodiazepine receptor changes. For every enzyme activity or binding property examined, no significant change was found when brains were kept at 4 degrees C for up to 72 h prior to freezing at -70 degrees C. Na,K-ATPase and low-affinity Ca-ATPase activities were also stable in brains kept at 25 degrees C for up to 72 h. Mg-ATPase activity was reduced in brains kept at 25 degrees C for 24 and 48 h. [3H]Guanidinoethylmercaptosuccinic acid [( 3H]GEMSA) binding to enkephalin convertase in the cytosol was not significantly changed in brains kept at 25 degrees C; however, a small increase was seen for [3H]GEMSA binding to the membrane fraction at 24, but not 48 and 72 h postmortem. [3H]Quinuclidinyl benzilate [( 3H]QNB) binding to muscarinic cholinergic receptors decreased in brains kept at 25 degrees C for 72 h. Opioid receptor binding also decreased in brains kept at 25 degrees C. Using [3H]2-D-alanine-5-D-leucine enkephalin to label delta opioid receptors, a statistically significant decrease in binding was observed as early as 6 h postmortem, and was completely abolished after 72 h at 25 degrees C. In contrast, [3H]naloxone binding was unchanged after 24 h at 25 degrees C, but was decreased after 48 and 72 h.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3029332

  20. Transport of membrane-bound mineral particles in blood vessels during chicken embryonic bone development.

    PubMed

    Kerschnitzki, Michael; Akiva, Anat; Ben Shoham, Adi; Koifman, Naama; Shimoni, Eyal; Rechav, Katya; Arraf, Alaa A; Schultheiss, Thomas M; Talmon, Yeshayahu; Zelzer, Elazar; Weiner, Stephen; Addadi, Lia

    2016-02-01

    During bone formation in embryos, large amounts of calcium and phosphate are taken up and transported to the site where solid mineral is first deposited. The initial mineral forms in vesicles inside osteoblasts and is deposited as a highly disordered calcium phosphate phase. The mineral is then translocated to the extracellular space where it penetrates the collagen matrix and crystallizes. To date little is known about the transport mechanisms of calcium and phosphate in the vascular system, especially when high transport rates are needed and the concentrations of these ions in the blood serum may exceed the solubility product of the mineral phase. Here we used a rapidly growing biological model, the chick embryo, to study the bone mineralization pathway taking advantage of the fact that large amounts of bone mineral constituents are transported. Cryo scanning electron microscopy together with cryo energy dispersive X-ray spectroscopy and focused-ion beam imaging in the serial surface view mode surprisingly reveal the presence of abundant vesicles containing small mineral particles in the lumen of the blood vessels. Morphologically similar vesicles are also found in the cells associated with bone formation. This observation directly implicates the vascular system in solid mineral distribution, as opposed to the transport of ions in solution. Mineral particle transport inside vesicles implies that far larger amounts of the bone mineral constituents can be transported through the vasculature, without the danger of ectopic precipitation. This introduces a new stage into the bone mineral formation pathway, with the first mineral being formed far from the bone itself. PMID:26481471

  1. Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen.

    PubMed

    Lopez, Jodie; Bittame, Amina; Massera, Céline; Vasseur, Virginie; Effantin, Grégory; Valat, Anne; Buaillon, Célia; Allart, Sophie; Fox, Barbara A; Rommereim, Leah M; Bzik, David J; Schoehn, Guy; Weissenhorn, Winfried; Dubremetz, Jean-François; Gagnon, Jean; Mercier, Corinne; Cesbron-Delauw, Marie-France; Blanchard, Nicolas

    2015-12-15

    Apicomplexa parasites such as Toxoplasma gondii target effectors to and across the boundary of their parasitophorous vacuole (PV), resulting in host cell subversion and potential presentation by MHC class I molecules for CD8 T cell recognition. The host-parasite interface comprises the PV limiting membrane and a highly curved, membranous intravacuolar network (IVN) of uncertain function. Here, using a cell-free minimal system, we dissect how membrane tubules are shaped by the parasite effectors GRA2 and GRA6. We show that membrane association regulates access of the GRA6 protective antigen to the MHC I pathway in infected cells. Although insertion of GRA6 in the PV membrane is key for immunogenicity, association of GRA6 with the IVN limits presentation and curtails GRA6-specific CD8 responses in mice. Thus, membrane deformations of the PV regulate access of antigens to the MHC class I pathway, and the IVN may play a role in immune modulation. PMID:26628378

  2. Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

    NASA Technical Reports Server (NTRS)

    Tischner, R.; Ward, M. R.; Huffaker, R. C.

    1989-01-01

    Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrate uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.

  3. Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane

    SciTech Connect

    Lieberman, R.L.; Rosenzweig, A.C.

    2010-03-08

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that catalyses the conversion of methane to methanol. Knowledge of how pMMO performs this extremely challenging chemistry may have an impact on the use of methane as an alternative energy source by facilitating the development of new synthetic catalysts. We have determined the structure of pMMO from the methanotroph Methylococcus capsulatus (Bath) to a resolution of 2.8 {angstrom}. The enzyme is a trimer with an {alpha}{sub 3}{beta}{sub 3}{gamma}{sub 3} polypeptide arrangement. Two metal centres, modelled as mononuclear copper and dinuclear copper, are located in soluble regions of each pmoB subunit, which resembles cytochrome c oxidase subunit II. A third metal centre, occupied by zinc in the crystal, is located within the membrane. The structure provides new insight into the molecular details of biological methane oxidation.

  4. Modulating role of 'Saptarangi' (Casearia esculenta) on membrane bound ATPase in streptozotocin diabetic rats.

    PubMed

    Prakasam, A; Sethupathy, S; Pugalendi, K V

    2005-11-01

    We have studied the activities of adenosine triphosphatase (Na+/K+ATPase, Mg2+ATPase, Ca2+ATPase and Total ATPase) in erythrocyte, liver, kidney and cardiac tissues of control and Casearia esculenta treated streptozotocin (STZ) diabetic rats. The activity of Na+/K+ATPase plays a central role in the regulation of intra and extra cellular homeostasis and alteration of this transport system is thought to be linked to several complications of diabetes mellitus. An Mg2+ dependent ATPase activity is responsible for controlling the energy requiring process in cells whereas Ca2+ATPase is responsible for the signal transduction pathways and membrane fluidity. Activities of these enzymes were significantly altered (p < 0.05) in STZ diabetic rats. Oral administration of C. esculenta root extract for a period of 45 days resulted in significant (p < 0.05) reversal of these enzymes' activities to near normal. Thus the results suggest that C. esculenta protects the membrane integrity and functional status in STZ diabetic rats. PMID:16320953

  5. Reconstitution of selective HIV-1 RNA packaging in vitro by membrane-bound Gag assemblies

    PubMed Central

    Carlson, Lars-Anders; Bai, Yun; Keane, Sarah C; Doudna, Jennifer A; Hurley, James H

    2016-01-01

    HIV-1 Gag selects and packages a dimeric, unspliced viral RNA in the context of a large excess of cytosolic human RNAs. As Gag assembles on the plasma membrane, the HIV-1 genome is enriched relative to cellular RNAs by an unknown mechanism. We used a minimal system consisting of purified RNAs, recombinant HIV-1 Gag and giant unilamellar vesicles to recapitulate the selective packaging of the 5’ untranslated region of the HIV-1 genome in the presence of excess competitor RNA. Mutations in the CA-CTD domain of Gag which subtly affect the self-assembly of Gag abrogated RNA selectivity. We further found that tRNA suppresses Gag membrane binding less when Gag has bound viral RNA. The ability of HIV-1 Gag to selectively package its RNA genome and its self-assembly on membranes are thus interdependent on one another. DOI: http://dx.doi.org/10.7554/eLife.14663.001 PMID:27343348

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

    PubMed Central

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

    1985-01-01

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

  7. The developmental regulation of the L2/HNK-1 and L3 carbohydrate epitopes in mouse brain. Evidence for separate control of lipid- and protein-bound epitopes.

    PubMed

    Breen, K C

    1989-04-10

    The carbohydrate epitopes L2/HNK-1 and L3 have previously been identified on various neural cell adhesion molecules and have been suggested to play a role in the mediation of cell-cell adhesion. In this study, the developmental expression of the two epitopes in soluble, membrane-bound and chloroform/methanol-extracted fractions of the constituent mouse brain regions was examined by enzyme-linked immunosorbent assay (ELISA). The protein-bound epitopes were shown to be uniformly developmentally regulated, with levels peaking at postnatal day 20 (P20). The epitopes in a crude chloroform/methanol fraction, however, demonstrated a different pattern, with L2 peaking earlier at postnatal day zero (P0). These results suggest a possible interaction between the control of the two pools of the epitope. PMID:2468531

  8. Cloning, purification, crystallization and preliminary X-ray analysis of the catalytic domain of human receptor-like protein tyrosine phosphatase [gamma] in three different crystal forms

    SciTech Connect

    Kish, Kevin; McDonnell, Patricia A.; Goldfarb, Valentina; Gao, Mian; Metzler, William J.; Langley, David R.; Bryson, James W.; Kiefer, Susan E.; Carpenter, Brian; Kostich, Walter A.; Westphal, Ryan S.; Sheriff, Steven

    2013-03-07

    Protein tyrosine phosphatase {gamma} is a membrane-bound receptor and is designated RPTP{gamma}. RPTP{gamma} and two mutants, RPTP{gamma}(V948I, S970T) and RPTP{gamma}(C858S, S970T), were recombinantly expressed and purified for X-ray crystallographic studies. The purified enzymes were crystallized using the hanging-drop vapor-diffusion method. Crystallographic data were obtained from several different crystal forms in the absence and the presence of inhibitor. In this paper, a description is given of how three different crystal forms were obtained that were used with various ligands. An orthorhombic crystal form and a trigonal crystal form were obtained both with and without ligand, and a monoclinic crystal form was only obtained in the presence of a particularly elaborated inhibitor.

  9. Assembling of Holotrichia parallela (dark black chafer) midgut tissue transcriptome and identification of midgut proteins that bind to Cry8Ea toxin from Bacillus thuringiensis.

    PubMed

    Shu, Changlong; Tan, Shuqian; Yin, Jiao; Soberón, Mario; Bravo, Alejandra; Liu, Chunqing; Geng, Lili; Song, Fuping; Li, Kebin; Zhang, Jie

    2015-09-01

    Holotrichia parallela is one of the most severe crop pests in China, affecting peanut, soybean, and sweet potato crops. Previous work showed that Cry8Ea toxin is highly effective against this insect. In order to identify Cry8Ea-binding proteins in the midgut cells of H. parallela larvae, we assembled a midgut tissue transcriptome by high-throughput sequencing and used this assembled transcriptome to identify Cry8Ea-binding proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, we obtained de novo sequences of cDNAs from midgut tissue of H. parallela larvae and used available cDNA data in the GenBank. In a parallel assay, we obtained 11 Cry8Ea-binding proteins by pull-down assays performed with midgut brush border membrane vesicles. Peptide sequences from these proteins were matched to the H. parallela newly assembled midgut transcriptome, and 10 proteins were identified. Some of the proteins were shown to be intracellular proteins forming part of the cell cytoskeleton and/or vesicle transport such as actin, myosin, clathrin, dynein, and tubulin among others. In addition, an apolipophorin, which is a protein involved in lipid metabolism, and a novel membrane-bound alanyl aminopeptidase were identified. Our results suggest that Cry8Ea-binding proteins could be different from those characterized for Cry1A toxins in lepidopteran insects. PMID:26135984

  10. Bis-Histidine-Coordinated Hemes in Four-Helix Bundles: How the Geometry of the Bundle Controls the Axial Imidazole Plane Orientations in Transmembrane Cytochromes of Mitochondrial Complexes II and III and Related Proteins

    PubMed Central

    Berry, Edward A.; Walker, F. Ann

    2009-01-01

    Early investigation of the EPR spectra of bis-histidine-coordinated membrane-bound ferriheme proteins led to the description of a spectral signal that had only one resolved feature. These became known as “highly anisotropic low-spin” (HALS) or “large gmax” ferriheme centers. Extensive work with small-molecule model heme complexes showed that this spectroscopic signature occurs in bis-imidazole ferrihemes in which the planes of the imidazole ligands are nearly perpendicular, Δϕ = 57–90°. In the last decade protein crystallographic studies have revealed the atomic structures of a number of examples of bis-histidine heme proteins. A frequent characteristic of these large gmax ferrihemes in membrane-bound proteins is the occurrence of the heme within a four-helix bundle with a left-handed twist. The histidine ligands occur at the same level on two diametrically opposed helices of the bundle. These ligands have the same side chain conformation and ligate heme iron on the bundle axis, resulting in a quasi-2-fold symmetric structure. The two non-ligand-bearing helices also obey this symmetry, and have a conserved small residue, usually glycine, where the edge of the heme ring makes contact with the helix backbones. In many cases this small residue is preceded by a threonine or serine residue whose side chain hydroxyl oxygen acts as a hydrogen-bond acceptor from the Nδ1 atom of the heme-ligating histidine. The Δϕ angle is thus determined by the common histidine side-chain conformation and the crossing angle of the ligand-bearing helices, in some cases constrained by H-bonds to the Ser/Thr residues on the non-ligand-bearing helices. PMID:18418633

  11. Phosphoregulatory protein 14-3-3 facilitates SAC1 transport from the endoplasmic reticulum

    PubMed Central

    Bajaj Pahuja, Kanika; Wang, Jinzhi; Blagoveshchenskaya, Anastasia; Lim, Lillian; Madhusudhan, M. S.; Mayinger, Peter; Schekman, Randy

    2015-01-01

    Most secretory cargo proteins in eukaryotes are synthesized in the endoplasmic reticulum and actively exported in membrane-bound vesicles that are formed by the cytosolic coat protein complex II (COPII). COPII proteins are assisted by a variety of cargo-specific adaptor proteins required for the concentration and export of secretory proteins from the endoplasmic reticulum (ER). Adaptor proteins are key regulators of cargo export, and defects in their function may result in disease phenotypes in mammals. Here we report the role of 14-3-3 proteins as a cytosolic adaptor in mediating SAC1 transport in COPII-coated vesicles. Sac1 is a phosphatidyl inositol-4 phosphate (PI4P) lipid phosphatase that undergoes serum dependent translocation between the endoplasmic reticulum and Golgi complex and controls cellular PI4P lipid levels. We developed a cell-free COPII vesicle budding reaction to examine SAC1 exit from the ER that requires COPII and at least one additional cytosolic factor, the 14-3-3 protein. Recombinant 14-3-3 protein stimulates the packaging of SAC1 into COPII vesicles and the sorting subunit of COPII, Sec24, interacts with 14-3-3. We identified a minimal sorting motif of SAC1 that is important for 14-3-3 binding and which controls SAC1 export from the ER. This LS motif is part of a 7-aa stretch, RLSNTSP, which is similar to the consensus 14-3-3 binding sequence. Homology models, based on the SAC1 structure from yeast, predict this region to be in the exposed exterior of the protein. Our data suggest a model in which the 14-3-3 protein mediates SAC1 traffic from the ER through direct interaction with a sorting signal and COPII. PMID:26056309

  12. Direct effects of ionizing radiation on integral membrane proteins. Noncovalent energy transfer requires specific interpeptide interactions

    SciTech Connect

    Jhun, E.; Jhun, B.H.; Jones, L.R.; Jung, C.Y. )

    1991-05-25

    The 12 transmembrane alpha helices (TMHs) of human erythrocyte glucose transporter were individually cut by pepsin digestion as membrane-bound 2.5-3.5-kDa peptide fragments. Radiation-induced chemical degradation of these fragments showed an average target size of 34 kDa. This is 10-12 x larger than the average size of an individual TMH, demonstrating that a significant energy transfer occurs among these TMHs in the absence of covalent linkage. Heating this TMH preparation at 100{degree}C for 15 min reduced the target size to 5 kDa or less, suggesting that the noncovalent energy transfer requires specific helix-helix interactions. Purified phospholamban, a small (6-kDa) integral membrane protein containing a single TMH, formed a pentameric assembly in sodium dodecyl sulfate. The chemical degradation target size of this phospholamban pentamer was 5-6 kDa, illustrating that not all integral membrane protein assemblies permit intersubunit energy transfer. These findings together with other published observations suggest strongly that significant noncovalent energy transfer can occur within the tertiary and quaternary structure of membrane proteins and that as yet undefined proper molecular interactions are required for such covalent energy transfer. Our results with pepsin-digested glucose transporter also illustrate the importance of the interhelical interaction as a predominating force in maintaining the tertiary structure of a transmembrane protein.

  13. Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1.

    PubMed

    Morgera, Francesca; Sallah, Margaret R; Dubuke, Michelle L; Gandhi, Pallavi; Brewer, Daniel N; Carr, Chavela M; Munson, Mary

    2012-01-01

    Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function-it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6-Sec1 interaction is exclusive of Sec6-Sec9 but compatible with Sec6-exocyst assembly. In contrast, the Sec6-exocyst interaction is incompatible with Sec6-Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6-exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion. PMID:22114349

  14. Degradation of gangliosides by the lysosomal sialidase requires an activator protein.

    PubMed

    Fingerhut, R; van der Horst, G T; Verheijen, F W; Conzelmann, E

    1992-09-15

    Lysosomal sialidase, which was formerly believed to degrade only water-soluble substrates but not glycolipids, cleaves ganglioside substrates II3NeuNAc-LacCer, IV3NeuNAc, II3NeuNAc-GgOse4Cer, IV3 NeuNAc, II3(NeuNAc)2-GgOse4Cer when these are dispersed either with an appropriate detergent (taurodeoxycholate) or with the sulfatide activator protein, a physiologic lipid solubilizer required for the lysosomal hydrolysis of other glycolipids by water-soluble hydrolases. In the presence of the activator protein, time and protein dependence were linear within wide limits, while the detergent rapidly inactivated the enzyme. The disialo group of the b-series gangliosides was only poorly attacked by the enzyme when the lipids were dispersed with the activator protein, whereas in the presence of the detergent, they were hydrolyzed as fast as terminal sialic acid residues. With the appropriate assay method, significant ganglioside sialidase activity could be demonstrated in the secondary lysosome fraction of normal skin fibroblasts but not of sialidosis fibroblasts. Our results support the notion that there is only one lysosomal sialidase, which degrades both the water-soluble and the membrane-bound sialyl glycoconjugates. PMID:1396669

  15. Alteration of cardiac glycoside positive inotropic action by modulators of protein synthesis and degradation

    SciTech Connect

    Nosek, T.M.; Adams, R.J.

    1986-03-05

    Numerous membrane bound and cytoplasmic proteins participate in the cardiac expression of the positive inotropic action (PIA) of digitalis glycosides including the Na,K-ATPase (NKA). Exposure of the myocardium to an inhibitor of protein synthesis (cycloheximide, CYC) or of protein degradation (leupeptin, LEU) alters the PIA of ouabain in isolated, paced guinea pig papillary muscles (PM) in opposite ways. In vivo exposure to CYC for 3 hr resulted in a 30% depression of the in vitro PIA of ouabain at 1.7..mu..M compared to control. In vivo exposure to LEU for 1 hr resulted in a 47% enhancement of the in vitro PIA of 1.7..mu..M ouabain. Neither drug had an apparent effect on the ouabain PIA ED50. Neither CYC nor LEU exposure to PM in vitro affect resting or developed tension or the response of skinned PM to calcium. The mechanisms of the PIA alterations by CYC or LEU do not involve a direct effect on the digitalis receptor. Exposure of isolated cardiac sarcolemma enriched in NKA to 10-100..mu..M CYC or LEU did not affect NKA activity or /sup 3/H-ouabain binding. Although direct physicochemical effects of CYC or LEU may be involved in the alterations of the ouabain PIA, it is possible that modulation of the cellular levels or turnover rate of short-lived proteins may affect cardiac regulation of the digitalis PIA.

  16. Construction of polycythemia vera protein interaction network and prediction of related biological functions.

    PubMed

    Liu, L-J; Cao, X-J; Zhou, C; Sun, Y; Lv, Q-L; Feng, F-B; Zhang, Y-Y; Sun, C-G

    2016-01-01

    Here, polycythemia vera (PV)-related genes were screened by the Online Mendelian Inheritance in Man (OMIM), and literature pertaining to the identified genes was extracted and a protein-protein interaction network was constructed using various Cytoscape plugins. Various molecular complexes were detected using the Clustervize plugin and a gene ontology-enrichment analysis of the biological pathways, molecular functions, and cellular components of the selected molecular complexes were identified using the BiNGo plugin. Fifty-four PV-related genes were identified in OMIM. The protein-protein interaction network contains 5 molecular complexes with correlation integral values >4. These complexes regulated various biological processes (peptide tyrosinase acidification, cell metabolism, and macromolecular biosynthesis), molecular functions (kinase activity, receptor binding, and cytokine activity), and the cellular components were mainly concentrated in the nucleus, intracellular membrane-bounded organelles, and extracellular region. These complexes were associated with the JAK-STAT signal transduction pathway, neurotrophic factor signaling pathway, and Wnt signaling pathway, which were correlated with chronic myeloid leukemia and acute myeloid leukemia. PMID:26909922

  17. A 42-kilodalton annexin-like protein is associated with plant vacuoles.

    PubMed Central

    Seals, D F; Parrish, M L; Randall, S K

    1994-01-01

    A 42-kD, calcium-dependent, membrane-binding protein (VCaB42) was associated with partially purified vacuole membrane. Membrane-dissociation assays indicated that VCaB42 binding to vacuole membranes was selective for calcium over other cations and that 50% of VCaB42 remained membrane bound at 61 +/- 11 nM free calcium. A 13-amino acid sequence obtained from VCaB42 showed 85% similarity with the endonexin fold, a sequence found in the annexin family of proteins that is thought to be essential for calcium and lipid binding. The greatest similarity in amino acid sequence was observed with annexin VIII (VAC-beta). The calcium-binding properties and sequence similarities suggest that VCaB42 is a member of the annexin family of calcium-dependent, membrane-binding proteins. Functional assays for VCaB42 on vacuole membrane transport processes indicated that it did not significantly affect the initial rate of calcium uptake into vacuole membrane vesicles. Because VCaB42 is vacuole localized (likely on the cytosolic surface of the vacuole) and is 50% dissociated within the physiological range of cytosolic free calcium, we hypothesize that this protein is a sensor that monitors cytosolic calcium levels and transmits that information to the vacuole. PMID:7846156

  18. Deleted in Malignant Brain Tumors-1 Protein (DMBT1): A Pattern Recognition Receptor with Multiple Binding Sites

    PubMed Central

    Ligtenberg, Antoon J. M.; Karlsson, Niclas G.; Veerman, Enno C. I.

    2010-01-01

    Deleted in Malignant Brain Tumors-1 protein (DMBT1), salivary agglutinin (DMBT1SAG), and lung glycoprotein-340 (DMBT1GP340) are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW). Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs. PMID:21614203

  19. Proteomic Analysis of Rhizoctonia solani Identifies Infection-specific, Redox Associated Proteins and Insight into Adaptation to Different Plant Hosts.

    PubMed

    Anderson, Jonathan P; Hane, James K; Stoll, Thomas; Pain, Nicholas; Hastie, Marcus L; Kaur, Parwinder; Hoogland, Christine; Gorman, Jeffrey J; Singh, Karam B

    2016-04-01

    Rhizoctonia solaniis an important root infecting pathogen of a range of food staples worldwide including wheat, rice, maize, soybean, potato and others. Conventional resistance breeding strategies are hindered by the absence of tractable genetic resistance in any crop host. Understanding the biology and pathogenicity mechanisms of this fungus is important for addressing these disease issues, however, little is known about howR. solanicauses disease. This study capitalizes on recent genomic studies by applying mass spectrometry based proteomics to identify soluble, membrane-bound and culture filtrate proteins produced under wheat infection and vegetative growth conditions. Many of the proteins found in the culture filtrate had predicted functions relating to modification of the plant cell wall, a major activity required for pathogenesis on the plant host, including a number found only under infection conditions. Other infection related proteins included a high proportion of proteins with redox associated functions and many novel proteins without functional classification. The majority of infection only proteins tested were confirmed to show transcript up-regulation during infection including a thaumatin which increased susceptibility toR. solaniwhen expressed inNicotiana benthamiana In addition, analysis of expression during infection of different plant hosts highlighted how the infection strategy of this broad host range pathogen can be adapted to the particular host being encountered. Data are available via ProteomeXchange with identifier PXD002806. PMID:26811357

  20. Proteomic Analysis of Rhizoctonia solani Identifies Infection-specific, Redox Associated Proteins and Insight into Adaptation to Different Plant Hosts*

    PubMed Central

    Anderson, Jonathan P.; Hane, James K.; Stoll, Thomas; Pain, Nicholas; Hastie, Marcus L.; Kaur, Parwinder; Hoogland, Christine; Gorman, Jeffrey J.; Singh, Karam B.

    2016-01-01

    Rhizoctonia solani is an important root infecting pathogen of a range of food staples worldwide including wheat, rice, maize, soybean, potato and others. Conventional resistance breeding strategies are hindered by the absence of tractable genetic resistance in any crop host. Understanding the biology and pathogenicity mechanisms of this fungus is important for addressing these disease issues, however, little is known about how R. solani causes disease. This study capitalizes on recent genomic studies by applying mass spectrometry based proteomics to identify soluble, membrane-bound and culture filtrate proteins produced under wheat infection and vegetative growth conditions. Many of the proteins found in the culture filtrate had predicted functions relating to modification of the plant cell wall, a major activity required for pathogenesis on the plant host, including a number found only under infection conditions. Other infection related proteins included a high proportion of proteins with redox associated functions and many novel proteins without functional classification. The majority of infection only proteins tested were confirmed to show transcript up-regulation during infection including a thaumatin which increased susceptibility to R. solani when expressed in Nicotiana benthamiana. In addition, analysis of expression during infection of different plant hosts highlighted how the infection strategy of this broad host range pathogen can be adapted to the particular host being encountered. Data are available via ProteomeXchange with identifier PXD002806. PMID:26811357

  1. Interaction of recombinant analogs of spider silk proteins 1F9 and 2E12 with phospholipid membranes.

    PubMed

    Antonenko, Yuri N; Perevoshchikova, Irina V; Davydova, Lyubov I; Agapov, Igor A; Bogush, Vladimir G

    2010-06-01

    Recombinant analogs of spider dragline silk proteins 1F9 and 2E12 are characterized by numerous repeats consisting of hydrophobic poly-Ala blocks and Gly-rich sequences with a substantial number of positively charged amino acid residues which suggest a pronounced ability to interact with negatively charged phospholipid membranes. Actually both proteins displayed substantial binding affinity towards lipid vesicles formed of acidic lipids as measured by fluorescence correlation spectroscopy (FCS) using rhodamine-labeled conjugates of the proteins. Both proteins did not induce liposome leakage, fusion or breakdown, but were able to bring about liposome aggregation. 1F9 was more active in the induction of liposome aggregation compared to 2E12. Interestingly, 2E12 markedly decreased the rate of calcium-induced liposome fusion. Circular dichroism data showed that binding of the proteins to negatively charged phosphatidylserine liposomes provoked transition from the left-handed helix of polyproline II (PPII) type to beta-structures and alpha-helices. The data suggested predominantly surface location of membrane bound proteins without significant perturbation of their hydrophobic core. PMID:20214876

  2. Heat stress activates the yeast high-osmolarity glycerol mitogen-activated protein kinase pathway, and protein tyrosine phosphatases are essential under heat stress.

    PubMed

    Winkler, Astrid; Arkind, Christopher; Mattison, Christopher P; Burkholder, Anne; Knoche, Kathryn; Ota, Irene

    2002-04-01

    The yeast high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway has been characterized as being activated solely by osmotic stress. In this work, we show that the Hog1 MAPK is also activated by heat stress and that Sho1, previously identified as a membrane-bound osmosensor, is required for heat stress activation of Hog1. The two-component signaling protein, Sln1, the second osmosensor in the HOG pathway, was not involved in heat stress activation of Hog1, suggesting that the Sho1 and Sln1 sensors discriminate between stresses. The possible function of Hog1 activation during heat stress was examined, and it was found that the hog1 delta strain does not recover as rapidly from heat stress as well as the wild type. It was also found that protein tyrosine phosphatases (PTPs) Ptp2 and Ptp3, which inactivate Hog1, have two functions during heat stress. First, they are essential for survival at elevated temperatures, preventing lethality due to Hog1 hyperactivation. Second, they block inappropriate cross talk between the HOG and the cell wall integrity MAPK pathways, suggesting that PTPs are important for maintaining specificity in MAPK signaling pathways. PMID:12455951

  3. Heat Stress Activates the Yeast High-Osmolarity Glycerol Mitogen-Activated Protein Kinase Pathway, and Protein Tyrosine Phosphatases Are Essential under Heat Stress

    PubMed Central

    Winkler, Astrid; Arkind, Christopher; Mattison, Christopher P.; Burkholder, Anne; Knoche, Kathryn; Ota, Irene

    2002-01-01

    The yeast high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway has been characterized as being activated solely by osmotic stress. In this work, we show that the Hog1 MAPK is also activated by heat stress and that Sho1, previously identified as a membrane-bound osmosensor, is required for heat stress activation of Hog1. The two-component signaling protein, Sln1, the second osmosensor in the HOG pathway, was not involved in heat stress activation of Hog1, suggesting that the Sho1 and Sln1 sensors discriminate between stresses. The possible function of Hog1 activation during heat stress was examined, and it was found that the hog1Δ strain does not recover as rapidly from heat stress as well as the wild type. It was also found that protein tyrosine phosphatases (PTPs) Ptp2 and Ptp3, which inactivate Hog1, have two functions during heat stress. First, they are essential for survival at elevated temperatures, preventing lethality due to Hog1 hyperactivation. Second, they block inappropriate cross talk between the HOG and the cell wall integrity MAPK pathways, suggesting that PTPs are important for maintaining specificity in MAPK signaling pathways. PMID:12455951

  4. A Drosophila protein family implicated in pheromone perception is related to Tay-Sachs GM2-activator protein.

    PubMed

    Starostina, Elena; Xu, Aiguo; Lin, Heping; Pikielny, Claudio W

    2009-01-01

    Low volatility, lipid-like cuticular hydrocarbon pheromones produced by Drosophila melanogaster females play an essential role in triggering and modulating mating behavior, but the chemosensory mechanisms involved remain poorly understood. Recently, we showed that the CheB42a protein, which is expressed in only 10 pheromone-sensing taste hairs on the front legs of males, modulates progression to late stages of male courtship behavior in response to female-specific cuticular hydrocarbons. Here we report that expression of all 12 genes in the CheB gene family is predominantly or exclusively gustatory-specific, and occurs in many different, often non-overlapping patterns. Only the Gr family of gustatory receptor genes displays a comparable variety of gustatory-specific expression patterns. Unlike Grs, however, expression of all but one CheB gene is sexually dimorphic. Like CheB42a, other CheBs may therefore function specifically in gustatory perception of pheromones. We also show that CheBs belong to the ML superfamily of lipid-binding proteins, and are most similar to human GM2-activator protein (GM2-AP). In particular, GM2-AP residues involved in ligand binding are conserved in CheBs but not in other ML proteins. Finally, CheB42a is specifically secreted into the inner lumen of pheromone-sensing taste hairs, where pheromones interact with membrane-bound receptors. We propose that CheB proteins interact directly with lipid-like Drosophila pheromones and modulate their detection by the gustatory signal transduction machinery. Furthermore, as loss of GM2-AP in Tay-Sachs disease prevents degradation of GM2 gangliosides and results in neurodegeneration, the function of CheBs in pheromone response may involve biochemical mechanisms critical for lipid metabolism in human neurons. PMID:18952610

  5. Enhancing protein stability by adsorption onto raftlike lipid domains.

    PubMed

    Litt, Jeffrey; Padala, Chakradhar; Asuri, Prashanth; Vutukuru, Srinavya; Athmakuri, Krishna; Kumar, Sanat; Dordick, Jonathan; Kane, Ravi S

    2009-05-27

    We demonstrate that the stability of adsorbed proteins can be enhanced by controlling the heterogeneity of the surfaceby creating raftlike domains in a soft liposomal membrane. Recent work has shown that enzymes adsorbed onto highly curved nanoscale supports can be more stable than those adsorbed on flat surfaces with nominally the same chemical structure. This effect has been attributed to a decrease in lateral interenzyme interactions on a curved surface. Exploiting this idea, we asked if adsorbing enzymes onto "patchy" surfaces composed of adsorbing and nonadsorbing regions can be used to reduce lateral interactions even on relatively flat surfaces. We demonstrate that creating domains on which an enzyme can adsorb enhances the stability of that enzyme under denaturing conditions. Furthermore, we demonstrate that the size of these domains has a considerable effect on the degree of stability imparted by adsorption. Such biomimetic raft-inspired systems may find use in applications ranging from biorecognition to the design of novel strategies for the separation of biomolecules and controlling the interaction of multicomponent membrane-bound enzymes. PMID:19385631

  6. Microchannel protein separation by electric field gradient focusing.

    PubMed

    Petsev, Dimiter N; Lopez, Gabriel P; Ivory, Cornelius F; Sibbett, Scott S

    2005-06-01

    A microchannel device is presented which separates and focuses charged proteins based on electric field gradient focusing. Separation is achieved by setting a constant electroosmotic flow velocity against step changes in electrophoretic velocity. Where these two velocities are balanced for a given analyte, the analyte focuses at that point because it is driven to it from all points within the channel. We demonstrate the separation and focusing of a binary mixture of bovine serum albumin and phycoerythrin. The device is constructed of intersecting microchannels in poly(dimethylsiloxane)(PDMS) inlaid with hollow dialysis fibers. The device uses no exotic chemicals such as antibodies or synthetic ampholytes, but operates instead by purely physical means involving the independent manipulation of electrophoretic and electroosmotic velocities. One important difference between this apparatus and most other devices designed for field-gradient focusing is the injection of current at discrete intersections in the channel rather than continuously along the length of a membrane-bound separation channel. PMID:15915250

  7. Microcompartments and protein machines in prokaryotes.

    PubMed

    Saier, Milton H

    2013-01-01

    The prokaryotic cell was once thought of as a 'bag of enzymes' with little or no intracellular compartmentalization. In this view, most reactions essential for life occurred as a consequence of random molecular collisions involving substrates, cofactors and cytoplasmic enzymes. Our current conception of a prokaryote is far from this view. We now consider a bacterium or an archaeon as a highly structured, nonrandom collection of functional membrane-embedded and proteinaceous molecular machines, each of which serves a specialized function. In this article we shall present an overview of such microcompartments including (1) the bacterial cytoskeleton and the apparati allowing DNA segregation during cell division; (2) energy transduction apparati involving light-driven proton pumping and ion gradient-driven ATP synthesis; (3) prokaryotic motility and taxis machines that mediate cell movements in response to gradients of chemicals and physical forces; (4) machines of protein folding, secretion and degradation; (5) metabolosomes carrying out specific chemical reactions; (6) 24-hour clocks allowing bacteria to coordinate their metabolic activities with the daily solar cycle, and (7) proteinaceous membrane compartmentalized structures such as sulfur granules and gas vacuoles. Membrane-bound prokaryotic organelles were considered in a recent Journal of Molecular Microbiology and Biotechnology written symposium concerned with membranous compartmentalization in bacteria [J Mol Microbiol Biotechnol 2013;23:1-192]. By contrast, in this symposium, we focus on proteinaceous microcompartments. These two symposia, taken together, provide the interested reader with an objective view of the remarkable complexity of what was once thought of as a simple noncompartmentalized cell. PMID:23920489

  8. Noncanonical Role for the Host Vps4 AAA+ ATPase ESCRT Protein in the Formation of Tomato Bushy Stunt Virus Replicase

    PubMed Central

    Pogany, Judit; Risco, Cristina; Nagy, Peter D.

    2014-01-01

    Assembling of the membrane-bound viral replicase complexes (VRCs) consisting of viral- and host-encoded proteins is a key step during the replication of positive-stranded RNA viruses in the infected cells. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host have revealed the involvement of eleven cellular ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. The ESCRT proteins are involved in endosomal sorting of cellular membrane proteins by forming multiprotein complexes, deforming membranes away from the cytosol and, ultimately, pinching off vesicles into the lumen of the endosomes. In this paper, we show an unexpected key role for the conserved Vps4p AAA+ ATPase, whose canonical function is to disassemble the ESCRT complexes and recycle them from the membranes back to the cytosol. We find that the tombusvirus p33 replication protein interacts with Vps4p and three ESCRT-III proteins. Interestingly, Vps4p is recruited to become a permanent component of the VRCs as shown by co-purification assays and immuno-EM. Vps4p is co-localized with the viral dsRNA and contacts the viral (+)RNA in the intracellular membrane. Deletion of Vps4p in yeast leads to the formation of crescent-like membrane structures instead of the characteristic spherule and vesicle-like structures. The in vitro assembled tombusvirus replicase based on cell-free extracts (CFE) from vps4Δ yeast is highly nuclease sensitive, in contrast with the nuclease insensitive replicase in wt CFE. These data suggest that the role of Vps4p and the ESCRT machinery is to aid building the membrane-bound VRCs, which become nuclease-insensitive to avoid the recognition by the host antiviral surveillance system and the destruction of the viral RNA. Other (+)RNA viruses of plants and animals might also subvert Vps4p and the ESCRT machinery for formation of VRCs, which require membrane deformation and spherule formation. PMID:24763736

  9. Glucagon induces disaggregation of polymer-like structures of the. alpha. subunit of the stimulatory G protein in liver membranes

    SciTech Connect

    Nakamura, Shunichi; Rodbell, M. )

    1991-08-15

    The hydrodynamic behavior of G{alpha}{sub s}, the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein (G protein), in octyl glucoside extracts of rat liver membranes was investigated. As was previously shown for G proteins similarly extracted from brain synaptoneurosomes, G{alpha}{sub s} behaved as polydisperse structures with S values higher than that of heterotrimeric G proteins. When G{alpha}{sub s} in its membrane-bound form was ({sup 32}P)ADP-ribosylated by cholera toxin and the treated membranes were extracted with octyl glucoside, > 35% of the labeled G{alpha}{sub s} was found in material that sedimented through sucrose gradients and contained relatively low levels of immunoreactive G{alpha}{sub s}. These finding suggest that the glucagon receptor selectivity interacts with polymer-like structures of G{alpha}{sub 2} and that activation by GTP({gamma}S) results in disaggregation. The role of the {beta} and {gamma} subunits of G proteins in the hormone-induced process is not clear since the polymer-like structures extracted with octyl glucoside are devoid of {beta} and {gamma} subunits.

  10. Seven Transmembrane Receptors as Shapeshifting Proteins: The Impact of Allosteric Modulation and Functional Selectivity on New Drug Discovery

    PubMed Central

    Miller, Laurence J.

    2010-01-01

    It is useful to consider seven transmembrane receptors (7TMRs) as disordered proteins able to allosterically respond to a number of binding partners. Considering 7TMRs as allosteric systems, affinity and efficacy can be thought of in terms of energy flow between a modulator, conduit (the receptor protein), and a number of guests. These guests can be other molecules, receptors, membrane-bound proteins, or signaling proteins in the cytosol. These vectorial flows of energy can yield standard canonical guest allostery (allosteric modification of drug effect), effects along the plane of the cell membrane (receptor oligomerization), or effects directed into the cytosol (differential signaling as functional selectivity). This review discusses these apparently diverse pharmacological effects in terms of molecular dynamics and protein ensemble theory, which tends to unify 7TMR behavior toward cells. Special consideration will be given to functional selectivity (biased agonism and biased antagonism) in terms of mechanism of action and potential therapeutic application. The explosion of technology that has enabled observation of diverse 7TMR behavior has also shown how drugs can have multiple (pluridimensional) efficacies and how this can cause paradoxical drug classification and nomenclatures. PMID:20392808

  11. The late chlamydial inclusion membrane is not derived from the endocytic pathway and is relatively deficient in host proteins.

    PubMed Central

    Taraska, T; Ward, D M; Ajioka, R S; Wyrick, P B; Davis-Kaplan, S R; Davis, C H; Kaplan, J

    1996-01-01

    Chlamydiae are obligate intracellular parasites which multiply within infected cells in a membrane-bound structure termed an inclusion. Newly internalized bacteria are surrounded by host plasma membrane; however, the source of membrane for the expansion of the inclusion is unknown. To determine if the membrane for the mature inclusion was derived by fusion with cellular organelles, we stained infected cells with fluorescent or electron-dense markers specific for organelles and examined inclusions for those markers. We observed no evidence for the presence of endoplasmic reticulum, Golgi, late endosomal, or lysosomal proteins in the inclusion. These data suggest that the expansion of the inclusion membrane, beginning 24 h postinoculation, does not occur by the addition of host proteins resulting from either de novo host synthesis or by fusion with preexisting membranes. To determine the source of the expanding inclusion membrane, antibodies were produced against isolated membranes from Chlamydia-infected mouse cells. The antibodies were demonstrated to be solely against Chlamydia-specified proteins by both immunoprecipitation of [35S]methionine-labeled extracts and Western blotting (immunoblotting). Techniques were used to semipermeabilize Chlamydia-infected cells without disrupting the permeability of the inclusion, allowing antibodies access to the outer surface of the inclusion membrane. Immunofluorescent staining demonstrated a ring-like fluorescence around inclusions in semipermeabilized cells, whereas Triton X-100-permeabilized cells showed staining throughout the inclusion. These studies demonstrate that the inclusion membrane is made up, in part, of Chlamydia-specified proteins and not of existing host membrane proteins. PMID:8751921

  12. Expression, characterisation and antigenicity of a truncated Hendra virus attachment protein expressed in the protozoan host Leishmania tarentolae.

    PubMed

    Fischer, Kerstin; dos Reis, Vinicius Pinho; Finke, Stefan; Sauerhering, Lucie; Stroh, Eileen; Karger, Axel; Maisner, Andrea; Groschup, Martin H; Diederich, Sandra; Balkema-Buschmann, Anne

    2016-02-01

    Hendra virus (HeV) is an emerging zoonotic paramyxovirus within the genus Henipavirus that has caused severe morbidity and mortality in humans and horses in Australia since 1994. HeV infection of host cells is mediated by the membrane bound attachment (G) and fusion (F) glycoproteins, that are essential for receptor binding and fusion of viral and cellular membranes. The eukaryotic unicellular parasite Leishmania tarentolae has recently been established as a powerful tool to express recombinant proteins with mammalian-like glycosylation patterns, but only few viral proteins have been expressed in this system so far. Here, we describe the purification of a truncated, Strep-tag labelled and soluble version of the HeV attachment protein (sHeV G) expressed in stably transfected L. tarentolae cells. After Strep-tag purification the identity of sHeV G was confirmed by immunoblotting and mass spectrometry. The functional binding of sHeV G to the HeV cell entry receptor ephrin-B2 was confirmed in several binding assays. Generated polyclonal rabbit antiserum against sHeV G reacted with both HeV and Nipah virus (NiV) G proteins in immunofluorescence assay and efficiently neutralised NiV infection, thus further supporting the preserved antigenicity of the purified protein. PMID:26585033

  13. Adaptor Protein Complex 2 (AP-2) Mediated, Clathrin Dependent Endocytosis, And Related Gene Activities, Are A Prominent Feature During Maturation Stage Amelogenesis

    PubMed Central

    LACRUZ, Rodrigo S.; BROOKES, Steven J.; WEN, Xin; JIMENEZ, Jaime M.; VIKMAN, Susanna; HU, Ping; WHITE, Shane N.; LYNGSTADAAS, S. Petter; OKAMOTO, Curtis T.; SMITH, Charles E.; PAINE, Michael L.

    2012-01-01

    Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real time PCR, we show that the expression of clathrin and adaptor protein subunits are up-regulated in maturation stage rodent enamel organ cells. AP-2 is the most up-regulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin dependent endocytosis, thus implying the likelihood of a specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also up-regulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1), cluster of differentiation 63 and 68 (Cd63 and Cd68), ATPase, H+ transporting, lysosomal V0 subunit D2 (Atp6v0d2), ATPase, H+ transporting, lysosomal V1 subunit B2 (Atp6v1b2), chloride channel, voltage-sensitive 7 (Clcn7) and cathepsin K (Ctsk). Immunohistological data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain® showed up-regulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor regulated pathway for the endocytosis of enamel matrix proteins. These data together

  14. Myristoylated. cap alpha. subunits of guanine nucleotide-binding regulatory proteins

    SciTech Connect

    Buss, J.E.; Mumby, S.M.; Casey, P.J.; Gilman, A.G.; Sefton, B.M.

    1987-11-01

    Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the ..cap alpha.. subunits of G/sub s/ (stimulatory) (..cap alpha../sub 45/ and ..cap alpha../sub 52/), a 41-kDa subunit of G/sub i/ (inhibitory) (..cap alpha../sub 41/), a 40-kDa protein (..cap alpha../sub 40/), and the 36-kDa ..beta.. subunit. No protein that comigrated with the ..cap alpha.. subunit of G/sup 0/ (unknown function) (..cap alpha../sub 39/) was detected. In cells grown in the presence of (/sup 3/H)myristic acid, ..cap alpha../sub 41/ and ..cap alpha../sub 40/ contained /sup 3/H label, while the ..beta.. subunit did not. Chemical analysis of lipids attached covalently to purified ..cap alpha../sub 41/ and ..cap alpha../sub 39/ from bovine brain also revealed myristic acid. Similar analysis of brain G protein ..beta.. and ..gamma.. subunits and of G/sub t/ (Transducin) subunits (..cap alpha.., ..beta.., and ..gamma..) failed to reveal fatty acids. The fatty acid associated with ..cap alpha../sub 41/ , ..cap alpha../sub 40/, and ..cap alpha../sub 39/ was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins.

  15. Expression and localization of predicted inclusion membrane proteins in Chlamydia trachomatis.

    PubMed

    Weber, Mary M; Bauler, Laura D; Lam, Jennifer; Hackstadt, Ted

    2015-12-01

    Chlamydia trachomatis is an obligate intracellular pathogen that replicates in a membrane-bound vacuole termed the inclusion. Early in the infection cycle, the pathogen extensively modifies the inclusion membrane through incorporation of numerous type III secreted effector proteins, called inclusion membrane proteins (Incs). These proteins are characterized by a bilobed hydrophobic domain of 40 amino acids. The presence of this domain has been used to predict up to 59 putative Incs for C. trachomatis; however, localization to the inclusion membrane with specific antibodies has been demonstrated for only about half of them. Here, we employed recently developed genetic tools to verify the localization of predicted Incs that had not been previously localized to the inclusion membrane. Expression of epitope-tagged putative Incs identified 10 that were previously unverified as inclusion membrane localized and thus authentic Incs. One novel Inc and 3 previously described Incs were localized to inclusion membrane microdomains, as evidenced by colocalization with phosphorylated Src (p-Src). Several predicted Incs did not localize to the inclusion membrane but instead remained associated with the bacteria. Using Yersinia as a surrogate host, we demonstrated that many of these are not secreted via type III secretion, further suggesting they may not be true Incs. Collectively, our results highlight the utility of genetic tools for demonstrating secretion from chlamydia. Further mechanistic studies aimed at elucidating effector function will advance our understanding of how the pathogen maintains its unique intracellular niche and mediates interactions with the host. PMID:26416906

  16. Congenital protein losing enteropathy: an inborn error of lipid metabolism due to DGAT1 mutations.

    PubMed

    Stephen, Joshi; Vilboux, Thierry; Haberman, Yael; Pri-Chen, Hadass; Pode-Shakked, Ben; Mazaheri, Sina; Marek-Yagel, Dina; Barel, Ortal; Di Segni, Ayelet; Eyal, Eran; Hout-Siloni, Goni; Lahad, Avishay; Shalem, Tzippora; Rechavi, Gideon; Malicdan, May Christine V; Weiss, Batia; Gahl, William A; Anikster, Yair

    2016-08-01

    Protein-losing enteropathy (PLE) is a clinical disorder of protein loss from the gastrointestinal system that results in hypoproteinemia and malnutrition. This condition is associated with a wide range of gastrointestinal disorders. Recently, a unique syndrome of congenital PLE associated with biallelic mutations in the DGAT1 gene has been reported in a single family. We hypothesize that mutations in this gene are responsible for undiagnosed cases of PLE in infancy. Here we investigated three children in two families presenting with severe diarrhea, hypoalbuminemia and PLE, using clinical studies, homozygosity mapping, and exome sequencing. In one family, homozygosity mapping using SNP arrays revealed the DGAT1 gene as the best candidate gene for the proband. Sequencing of all the exons including flanking regions and promoter regions of the gene identified a novel homozygous missense variant, p.(Leu295Pro), in the highly conserved membrane-bound O-acyl transferase (MBOAT) domain of the DGAT1 protein. Expression studies verified reduced amounts of DGAT1 in patient fibroblasts. In a second family, exome sequencing identified a previously reported splice site mutation in intron 8. These cases of DGAT1 deficiency extend the molecular and phenotypic spectrum of PLE, suggesting a re-evaluation of the use of DGAT1 inhibitors for metabolic disorders including obesity and diabetes. PMID:26883093

  17. DELLA proteins regulate expression of a subset of AM symbiosis-induced genes in Medicago truncatula.

    PubMed

    Floss, Daniela S; Lévesque-Tremblay, Véronique; Park, Hee-Jin; Harrison, Maria J

    2016-04-01

    The majority of the vascular flowering plants form symbiotic associations with fungi from the phylum Glomeromycota through which both partners gain access to nutrients, either mineral nutrients in the case of the plant, or carbon, in the case of the fungus. (1) The association develops in the roots and requires substantial remodeling of the root cortical cells where branched fungal hyphae, called arbuscules, are housed in a new membrane-bound apoplastic compartment. (2) Nutrient exchange between the symbionts occurs over this interface and its development and maintenance is critical for symbiosis. Previously, we showed that DELLA proteins, which are well known as repressors of gibberellic acid signaling, also regulate development of AM symbiosis and are necessary to enable arbuscule development. (3) Furthermore, constitutive overexpression of a dominant DELLA protein (della1-Δ18) is sufficient to induce transcripts of several AM symbiosis-induced genes, even in the absence of the fungal symbiont. (4) Here we further extend this approach and identify AM symbiosis genes that respond transcriptionally to constitutive expression of a dominant DELLA protein and also genes that do respond to this treatment. Additionally, we demonstrate that DELLAs interact with REQUIRED FOR ARBUSCULE DEVELOPMENT 1 (RAD1) which further extends our knowledge of GRAS factor complexes that have the potential to regulate gene expression during AM symbiosis. PMID:26984507

  18. Probing Polytopic Membrane Protein-Substrate Interactions by Luminescence Resonance Energy Transfer.

    PubMed

    Musial-Siwek, Monika; Jaffee, Marcie B; Imperiali, Barbara

    2016-03-23

    Integral membrane proteins play essential roles in all living systems; however, major technical hurdles challenge analyses of this class of proteins. Biophysical approaches that provide structural information to complement and leverage experimentally determined and computationally predicted structures are urgently needed. Herein we present the application of luminescence resonance energy transfer (LRET) for investigating the interactions of the polytopic membrane-bound oligosaccharyl transferases (OTases) with partner substrates. Monomeric OTases, such as the PglBs from Campylobacter jejuni and Campylobacter lari, catalyze transfer of glycans from membrane-associated undecaprenol diphosphate-linked substrates to proteins in the bacterial periplasm. LRET-based distance measurements are enabled by the inclusion of an encoded N-terminal lanthanide-binding tag (LBT), and LRET between the luminescent (LBT)-Tb(3+) donor complex and fluorescently labeled peptide and glycan substrates provides discrete distance measurements across the span of the membrane. LRET-based measurements of detergent-solubilized PglB from C. lari allowed direct comparison with the distances based on the previously reported the C. lari PglB crystal structure, thereby validating the approach in a defined system. Distance measurements between peptide and glycan substrates and the C. jejuni PglB offer new experimental information on substrate binding to the related, but structurally uncharacterized, eukaryotic OTase. PMID:26918528

  19. Solution structure of an EGF module pair from the Plasmodium falciparum merozoite surface protein 1.

    PubMed

    Morgan, W D; Birdsall, B; Frenkiel, T A; Gradwell, M G; Burghaus, P A; Syed, S E; Uthaipibull, C; Holder, A A; Feeney, J

    1999-05-28

    The solution structure of the 96-residue C-terminal fragment of the merozoite surface protein 1 (MSP-1) from Plasmodium falciparum has been determined using nuclear magnetic resonance (NMR) spectroscopic measurements on uniformly13C/15N-labelled protein, efficiently expressed in the methylotrophic yeast Komagataella (Pichia) pastoris. The structure has two domains with epidermal growth factor (EGF)-like folds with a novel domain interface for the EGF domain pair interactions, formed from a cluster of hydrophobic residues. This gives the protein a U-shaped overall structure with the N-terminal proteolytic processing site close to the C-terminal glycosyl phosphatidyl inositol (GPI) membrane anchor site, which is consistent with the involvement of a membrane-bound proteinase in the processing of MSP-1 during erythrocyte invasion. This structure, which is the first protozoan EGF example to be determined, contrasts with the elongated structures seen for EGF-module pairs having shared Ca2+-ligation sites at their interface, as found, for example, in fibrillin-1. Recognition surfaces for antibodies that inhibit processing and invasion, and antibodies that block the binding of these inhibitory antibodies, have been mapped on the three-dimensional structure by considering specific MSP-1 mutants. PMID:10339410

  20. Ebola virus glycoprotein Fc fusion protein confers protection against lethal challenge in vaccinated mice

    PubMed Central

    Konduru, Krishnamurthy; Bradfute, Steven B.; Jacques, Jerome; Manangeeswaran, Mohanraj; Nakamura, Siham; Morshed, Sufi; Wood, Steven C.; Bavari, Sina

    2011-01-01

    Ebola virus is a Filoviridae that causes hemorrhagic fever in humans and induces high morbidity and mortality rates. Filoviruses are classified as "Category A bioterrorism agents", and currently there are no licensed therapeutics or vaccines to treat and prevent infection. The Filovirus glycoprotein (GP) is sufficient to protect individuals against infection, and several vaccines based on GP are under development including recombinant adenovirus, parainfluenza virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus (VSV) and virus-like particles. Here we describe the development of a GP Fc fusion protein as a vaccine candidate. We expressed the extracellular domain of the Zaire Ebola virus (ZEBOV) GP fused to the Fc fragment of human IgG1 (ZEBOVGP-Fc) in mammalian cells and showed that GP undergoes the complex furin cleavage and processing observed in the native membrane-bound GP. Mice immunized with ZEBOVGP-Fc developed T-cell immunity against ZEBOV GP and neutralizing antibodies against replication-competent VSV-G deleted recombinant VSV containing ZEBOV GP. The ZEBOVGP-Fc vaccinated mice were protected against challenge with a lethal dose of ZEBOV. These results show that vaccination with the ZEBOVGP-Fc fusion protein alone without the need of a viral vector or assembly into virus-like particles is sufficient to induce protective immunity against ZEBOV in mice. Our data suggested that Filovirus GP Fc fusion proteins could be developed as a simple, safe, efficacious, and cost effective vaccine against Filovirus infection for human use. PMID:21329775

  1. Specific changes in rapidly transported proteins during regeneration of the goldfish optic nerve

    SciTech Connect

    Benowitz, L.I.; Shashoua, V.E.; Yoon, M.G.

    1981-03-01

    Double labeling methods were used to identify changes in the complement of proteins synthesized in the retinal ganglion cells and transported down the optic nerve during the process of axonal regeneration. Eight to 62 days after goldfish underwent a unilateral optic nerve crush, one eye was labeled with (3H)-, the other with (14C)proline. Control and regenerating optic nerves were dissected out and homogenized together after 5 hr, a time which allowed us to examine selectively membrane-bound components which migrate in the rapid phase of axoplasmic transport. Proteins from the two sides were so-purified and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the 3H and 14C incorporation patterns along the gels revealed a radical shift away from the normal labeling spectrum during regeneration, with selective changes in labeling at particular molecular weights varying over a 3-fold range. Eight days after crushing the optic nerve, the greatest increases in labeling were seen for material with apparent molecular weights of 24,000 to 27,000, 44,000, and 210,000 daltons. These peaks declined thereafter, and on days 29 to 39, the most prominent increases were at 110,000 to 140,000 daltons. These studies indicate a continuously changing pattern in the synthesis and/or degradation of proteins that are rapidly transported down the optic nerve during regeneration and point to molecular species potential significance in the establishment of the visual map upon the brain.

  2. GPI transamidase and GPI anchored proteins: oncogenes and biomarkers for cancer.

    PubMed

    Gamage, Dilani G; Hendrickson, Tamara L

    2013-01-01

    Cancer is second only to heart disease as a cause of death in the US, with a further negative economic impact on society. Over the past decade, details have emerged which suggest that different glycosylphosphatidylinositol (GPI)-anchored proteins are fundamentally involved in a range of cancers. This post-translational glycolipid modification is introduced into proteins via the action of the enzyme GPI transamidase (GPI-T). In 2004, PIG-U, one of the subunits of GPI-T, was identified as an oncogene in bladder cancer, offering a direct connection between GPI-T and cancer. GPI-T is a membrane-bound, multi-subunit enzyme that is poorly understood, due to its structural complexity and membrane solubility. This review is divided into three sections. First, we describe our current understanding of GPI-T, including what is known about each subunit and their roles in the GPI-T reaction. Next, we review the literature connecting GPI-T to different cancers with an emphasis on the variations in GPI-T subunit over-expression. Finally, we discuss some of the GPI-anchored proteins known to be involved in cancer onset and progression and that serve as potential biomarkers for disease-selective therapies. Given that functions for only one of GPI-T's subunits have been robustly assigned, the separation between healthy and malignant GPI-T activity is poorly defined. PMID:23978072

  3. Zinc ions and alkaline pH alter the phosphorylation state of human erythrocyte membrane proteins

    SciTech Connect

    Fennell, R.L. Jr.

    1988-01-01

    Since the phosphorylation state of the red cell membrane proteins in vitro is likely to be regulated by phosphorylation and dephosphorylation, this research was carried out to investigate the possible role of membrane-bound phosphatase activities. These studies were conducted with red blood cell ghosts and IOVs from normal individuals and from an individual with hereditary spherocytosis. In vitro phosphorylation with ({gamma}-{sup 32}P) ATP was conducted in the presence and the absence of Zn{sup ++}, or erythrocyte ghosts and IOVs were pretreated for 30 minutes at 37{degree}C and pH 7-11 in the presence and the absence of calf intestine alkaline phosphatase. The resulting phosphoproteins were analyzed by SDS-polyacrylamide gel electrophoresis, stained with Coomassie blue, and fluorographed. In the presence of Zn{sup ++}, the red blood ghosts, with or without pretreatment, demonstrated enhanced phosphorylation of membrane proteins, including band 4.2. Preincubation at pH 10 in the presence of absence of exogenous phosphatase further stimulates phosphorylation of these proteins. Under similar conditions, the erythrocyte membranes also demonstrated the ability to hydrolyze p-nitrophenyl phosphate and to remove {sup 32}P from red blood cell phosphoproteins.

  4. Basement membrane protein nidogen-1 is a target of meprin β in cisplatin nephrotoxicity

    PubMed Central

    Herzog, Christian; Marisiddaiah, Raju; Haun, Randy S.; Kaushal, Gur P.

    2015-01-01

    Meprins are oligomeric metalloproteinases that are abundantly expressed in the brush-border membranes of renal proximal tubules. During acute kidney injury (AKI) induced by cisplatin or ischemia-reperfusion, membrane-bound meprins are shed and their localization is altered from the apical membranes toward the basolateral surface of the proximal tubules. Meprins are capable of cleaving basement membrane proteins in vitro, however, it is not known whether meprins are able of degrade extracellular matrix proteins under pathophysiological conditions in vivo. The present study demonstrates that a basement membrane protein, nidogen-1, is cleaved and excreted in the urine of mice subjected to cisplatin-induced nephrotoxicity, a model of AKI. Cleaved nidogen-1 was not detected in the urine of untreated mice, but during the progression of cisplatin nephrotoxicity, the excretion of cleaved nidogen-1 increased in a time-dependent manner. The meprin inhibitor actinonin markedly prevented urinary excretion of the cleaved nidogen-1. In addition, meprin β-deficient mice, but not meprin α-deficient mice, subjected to cisplatin nephrotoxicity significantly suppressed excretion of cleaved nidogen-1, further suggesting that meprin β is involved in the cleavage of nidogen-1. These studies provide strong evidence for a pathophysiological link between meprin β and urinary excretion of cleaved nidogen-1 during cisplatin-induced AKI. PMID:25957482

  5. 1,2-diacylglycerols, but not phorbol esters, activate a potential inhibitory pathway for protein kinase C in GH/sub 3/ pituitary cells

    SciTech Connect

    Kolesnick, R.N.; Clegg, S.

    1988-05-15

    It has been suggested that sphingoid bases may serve as physiologic inhibitors of protein kinase C. Because 1,2-diacylglycerols, but not phorbol esters, enhance sphingomyelin degradation via a sphingomyelinase in GH/sub 3/ pituitary cells, the effects of phorbol esters, 1,2-diacylglycerols, and sphingomyelinase on protein kinase C activation were assessed. Under basal conditions, the inactive cytosolic form of protein kinase C predominated. 1,2-Diacylglycerols stimulated transient protein kinase C redistribution to the membrane. 1.2-Dioctanoylglycerol (200 ..mu..g/ml) reduced cytosolic protein kinase C activity to 67% of control. In contrast, the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated redistribution to the membrane without return to the cytosol. Exogenous sphingomyelinase reduced membrane-bound protein kinase C activity to 30% of control, yet did not alter cytosolic activity. Sphingomyelinase, added after phorbol ester-induced redistribution was completed, restored activity to the cytosol. These studies suggest that a pathway involving a sphingomyelinase might comprise a physiologic negative effector system for protein kinase C. Further, the failure of phorbol esters to activate this system might account for some differences between these agents.

  6. Purification of the extracellular domain of the membrane protein GlialCAM expressed in HEK and CHO cells and comparison of the glycosylation.

    PubMed

    Gaudry, Jean-Philippe; Arod, Christian; Sauvage, Christophe; Busso, Stephane; Dupraz, Philippe; Pankiewicz, Renata; Antonsson, Bruno

    2008-03-01

    Adhesion molecules are essential for a wide range of biological and physiological functions, including cell-cell interactions, cell interactions with the extracellular matrix, cell migration, proliferation and survival. Defects in cell adhesion have been associated with pathological conditions such as neoplasia, and neurodegenerative diseases. We have identified a new adhesion molecule of the immunoglobulin family, GlialCAM. The same protein was recently published under the name hepaCAM and was suggested to be associated with hepatocellular carcinoma. Here we have expressed and purified the extracellular domain of this molecule in two mammalian expression systems, HEK and CHO cells. A three step purification protocol gave an over 95% pure protein. The extracellular domain of GlialCAM possesses several potential N- and O-glycosylation sites. Glycosylation is one of the most common post-translational modifications of secreted proteins and of the extracellular domains of membrane bound proteins. It can influence both the activity and the stability of the protein. The glycosylation pattern has been shown to depend on the cell type where the protein is expressed. We examined if differences in the glycosylation of this protein could be detected when it was expressed in the two commonly used mammalian expression systems, HEK and CHO. Differences in the glycosylation were detected. PMID:18082421

  7. The Unfolded Protein Response Element IRE1α Senses Bacterial Proteins Invading the ER to Activate RIG-I and Innate Immune Signaling

    PubMed Central

    Cho, Jin A.; Lee, Ann-Hwee; Platzer, Barbara; Cross, Benedict C.S.; Gardner, Brooke M.; De Luca, Heidi; Luong, Phi; Harding, Heather P.; Glimcher, Laurie H.; Walter, Peter; Fiebiger, Edda; Ron, David; Kagan, Jonathan C.; Lencer, Wayne I.

    2013-01-01

    SUMMARY The plasma membrane and all membrane-bound organelles except for the Golgi and endoplasmic reticulum (ER) are equipped with pattern-recognition molecules to sense microbes or their products and induce innate immunity for host defense. Here, we report that inositol-requiring-1α (IRE1α), an ER protein that signals in the unfolded protein response (UPR), is activated to induce inflammation by binding a portion of cholera toxin as it co-opts the ER to cause disease. Other known UPR transducers, including the IRE1α-dependent transcription factor XBP1, are dispensable for this signaling. The inflammatory response depends instead on the RNase activity of IRE1α to degrade endogenous mRNA, a process termed regulated IRE1α-dependent decay (RIDD) of mRNA. The mRNA fragments produced engage retinoic-acid inducible gene 1 (RIG-I), a cyto-solic sensor of RNA viruses, to activate NF-κB and interferon pathways. We propose IRE1α provides for a generalized mechanism of innate immune surveillance originating within the ER lumen. PMID:23684307

  8. Residues in the alpha helix 7 of the bacterial maltose binding protein which are important in interactions with the Mal FGK2 complex.

    PubMed

    Szmelcman, S; Sassoon, N; Hofnung, M

    1997-03-01

    The periplasmic maltose binding protein, MalE, is a major element in maltose transport and in chemotaxis towards this sugar. Previous genetic analysis of the MalE protein revealed functional domains involved in transport and chemotactic functions. Among them the surface located alpha helix 7, which is part of the C-lobe, one of the two lobes forming the three dimensional structure of MalE. Small deletions in this region abolished maltose transport, although maintaining wild-type affinity and specificity as well as a normal chemoreceptor function. It was suggested that alpha helix 7 may be implicated in interactions between the maltose binding protein and the membrane-bound protein complex (Duplay P, Szmelcman S. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. II. Chemotaxis towards maltose. J Mol Biol 194:675-678: Duplay P, Szmelcman S, Bedouelle H, Hofnung M. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. I: Transport of maltose. J Mol Biol 194:663-673). In this study, we submitted a region of 14 residues--Asp 207 to Gly 220--encompassing alpha helix 7, to genetic analysis by oligonucleotide mediated random mutagenesis. Out of 127 identified mutations, twelve single and five double mutants with normal affinities towards maltose were selected for further investigation. Two types of mutations were characterized, silent mutations that did not affect maltose transport and mutations that heavily impaired transport kinetics, even thought the maltose binding capacity of the mutant proteins remained normal. Three substitutions at Tyr 210 (Y210S, Y210L, Y210N) drastically reduced maltose transport. One substitution at Ala 213 (A213I) and one substitution at Glu 214 (E214K) also impaired transport. These three identified residues, Tyr 210, Ala 213, and Glu 214, which are constituents of alpha helix 7, therefore seem to play some important role in maltose

  9. Quantitative Liver-Specific Protein Fingerprint in Blood: A Signature for Hepatotoxicity

    PubMed Central

    Hu, Zhiyuan; Lausted, Christopher; Yoo, Hyuntae; Yan, Xiaowei; Brightman, Amy; Chen, Jiankui; Wang, Weizhi; Bu, Xiangli; Hood, Leroy

    2014-01-01

    We discuss here a new approach to detecting hepatotoxicity by employing concentration changes of liver-specific blood proteins during disease progression. These proteins are capable of assessing the behaviors of their cognate liver biological networks for toxicity or disease perturbations. Blood biomarkers are highly desirable diagnostics as blood is easily accessible and baths virtually all organs. Fifteen liver-specific blood proteins were identified as markers of acetaminophen (APAP)-induced hepatotoxicity using three proteomic technologies: label-free antibody microarrays, quantitative immunoblotting, and targeted iTRAQ mass spectrometry. Liver-specific blood proteins produced a toxicity signature of eleven elevated and four attenuated blood protein levels. These blood protein perturbations begin to provide a systems view of key mechanistic features of APAP-induced liver injury relating to glutathione and S-adenosyl-L-methionine (SAMe) depletion, mitochondrial dysfunction, and liver responses to the stress. Two markers, elevated membrane-bound catechol-O-methyltransferase (MB-COMT) and attenuated retinol binding protein 4 (RBP4), report hepatic injury significantly earlier than the current gold standard liver biomarker, alanine transaminase (ALT). These biomarkers were perturbed prior to onset of irreversible liver injury. Ideal markers should be applicable for both rodent model studies and human clinical trials. Five of these mouse liver-specific blood markers had human orthologs that were also found to be responsive to human hepatotoxicity. This panel of liver-specific proteins has the potential to effectively identify the early toxicity onset, the nature and extent of liver injury and report on some of the APAP-perturbed liver networks. PMID:24465277

  10. The biological activities of protein/oleic acid complexes reside in the fatty acid.

    PubMed

    Fontana, Angelo; Spolaore, Barbara; Polverino de Laureto, Patrizia

    2013-06-01

    A complex formed by human α-lactalbumin (α-LA) and oleic acid (OA), named HAMLET, has been shown to have an apoptotic activity leading to the selective death of tumor cells. In numerous publications it has been reported that in the complex α-LA is monomeric and adopts a partly folded or "molten globule" state, leading to the idea that partly folded proteins can have "beneficial effects". The protein/OA molar ratio initially has been reported to be 1:1, while recent data have indicated that the OA-complex is given by an oligomeric protein capable of binding numerous OA molecules per protein monomer. Proteolytic fragments of α-LA, as well as other proteins unrelated to α-LA, can form OA-complexes with biological activities similar to those of HAMLET, thus indicating that a generic protein can form a cytotoxic complex under suitable experimental conditions. Moreover, even the selective tumoricidal activity of HAMLET-like complexes has been questioned. There is recent evidence that the biological activity of long chain unsaturated fatty acids, including OA, can be ascribed to their effect of perturbing the structure of biological membranes and consequently the function of membrane-bound proteins. In general, it has been observed that the cytotoxic effects exerted by HAMLET-like complexes are similar to those reported for OA alone. Overall, these findings can be interpreted by considering that the protein moiety does not have a toxic effect on its own, but merely acts as a solubilising agent for the inherently toxic fatty acid. PMID:23499846

  11. Unique apicomplexan IMC sub-compartment proteins are early markers for apical polarity in the malaria parasite

    PubMed Central

    Poulin, Benoit; Patzewitz, Eva-Maria; Brady, Declan; Silvie, Olivier; Wright, Megan H.; Ferguson, David J. P.; Wall, Richard J.; Whipple, Sarah; Guttery, David S.; Tate, Edward W.; Wickstead, Bill; Holder, Anthony A.; Tewari, Rita

    2013-01-01

    Summary The phylum Apicomplexa comprises over 5000 intracellular protozoan parasites, including Plasmodium and Toxoplasma, that are clinically important pathogens affecting humans and livestock. Malaria parasites belonging to the genus Plasmodium possess a pellicle comprised of a plasmalemma and inner membrane complex (IMC), which is implicated in parasite motility and invasion. Using live cell imaging and reverse genetics in the rodent malaria model P. berghei, we localise two unique IMC sub-compartment proteins (ISPs) and examine their role in defining apical polarity during zygote (ookinete) development. We show that these proteins localise to the anterior apical end of the parasite where IMC organisation is initiated, and are expressed at all developmental stages, especially those that are invasive. Both ISP proteins are N-myristoylated, phosphorylated and membrane-bound. Gene disruption studies suggest that ISP1 is likely essential for parasite development, whereas ISP3 is not. However, an absence of ISP3 alters the apical localisation of ISP1 in all invasive stages including ookinetes and sporozoites, suggesting a coordinated function for these proteins in the organisation of apical polarity in the parasite. PMID:24244852

  12. Effects of macromolecular crowding on a small lipid binding protein probed at the single-amino acid level.

    PubMed

    Pérez Santero, Silvia; Favretto, Filippo; Zanzoni, Serena; Chignola, Roberto; Assfalg, Michael; D'Onofrio, Mariapina

    2016-09-15

    Macromolecular crowding is a distinctive feature of the cellular interior, influencing the behaviour of biomacromolecules. Despite significant advancements in the description of the effects of crowding on global protein properties, the influence of cellular components on local protein attributes has received limited attention. Here, we describe a residue-level systematic interrogation of the structural, dynamic, and binding properties of the liver fatty acid binding protein (LFABP) in crowded solutions. Two-dimensional NMR spectral fingerprints and relaxation data were collected on LFABP in the presence of polymeric and biomolecular crowders. Non-interacting crowders produced minimal site-specific spectral perturbations on ligand-free and lipid-bound LFABP. Conformational adaptations upon ligand binding reproduced those observed in dilute solution, but a perturbation of the free oleate state resulted in less favorable uptake. When LFABP engaged in direct interactions with background molecules, changes in local chemical environments were detected for residues of the internal binding pocket and of the external surface. Enhanced complexity was introduced by investigating LFABP in cell lysates, and in membrane-bounded compartments. LFABP was able to capture ligands from prokaryotic and eukaryotic cell lysates, and from artificial cells (water-in-oil emulsion droplets). The data suggest that promiscuous interactions are a major factor influencing protein function in the cell. PMID:27457417

  13. Cholesterol Promotes Interaction of the Protein CLIC1 with Phospholipid Monolayers at the Air-Water Interface.

    PubMed

    Hossain, Khondker R; Al Khamici, Heba; Holt, Stephen A; Valenzuela, Stella M

    2016-01-01

    CLIC1 is a Chloride Intracellular Ion Channel protein that exists either in a soluble state in the cytoplasm or as a membrane bound protein. Members of the CLIC family are largely soluble proteins that possess the intriguing property of spontaneous insertion into phospholipid bilayers to form integral membrane ion channels. The regulatory role of cholesterol in the ion-channel activity of CLIC1 in tethered lipid bilayers was previously assessed using impedance spectroscopy. Here we extend this investigation by evaluating the influence of cholesterol on the spontaneous membrane insertion of CLIC1 into Langmuir film monolayers prepared using 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine alone or in combination with cholesterol. The spontaneous membrane insertion of CLIC1 was shown to be dependent on the presence of cholesterol in the membrane. Furthermore, pre-incubation of CLIC1 with cholesterol prior to its addition to the Langmuir film, showed no membrane insertion even in monolayers containing cholesterol, suggesting the formation of a CLIC1-cholesterol pre-complex. Our results therefore suggest that CLIC1 membrane interaction involves CLIC1 binding to cholesterol located in the membrane for its initial docking followed by insertion. Subsequent structural rearrangements of the protein would likely also be required along with oligomerisation to form functional ion channels. PMID:26875987

  14. Microsecond atomic force sensing of protein conformational dynamics: Implications for the primary light-induced events in bacteriorhodopsin

    PubMed Central

    Rousso, Itay; Khachatryan, Edward; Gat, Yahaloma; Brodsky, Igor; Ottolenghi, Michael; Sheves, Mordechai; Lewis, Aaron

    1997-01-01

    In this paper a new atomic force sensing technique is presented for dynamically probing conformational changes in proteins. The method is applied to the light-induced changes in the membrane-bound proton pump bacteriorhodopsin (bR). The microsecond time-resolution of the method, as presently implemented, covers many of the intermediates of the bR photocycle which is well characterized by spectroscopical methods. In addition to the native pigment, we have studied bR proteins substituted with chemically modified retinal chromophores. These synthetic chromophores were designed to restrict their ability to isomerize, while maintaining the basic characteristic of a large light-induced charge redistribution in the vertically excited Franck–Condon state. An analysis of the atomic force sensing signals lead us to conclude that protein conformational changes in bR can be init