FMP Publications

Our publications are recorded in a searchable database since 2010, updates will be added regularly.

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Different intra- and intermolecular activation mechanisms at the human lutropin receptor: Lutropin induces only cis- and choriogonadotropin also trans-activation
Grzesik, P., Teichmann, A., Furkert, J., Rutz, C., Wiesner, B., Kleinau(*), G., Schülein, R., Gromoll(*), J.; Krause, G.
Exp Clin Endocr Diab, 121

Tags: Structural Bioinformatics and Protein Design (Krause, G.), Cellular Imaging (Wiesner), Protein Trafficking (Schülein)

In tight junctions, claudins regulate the interactions between occludin, tricellulin and marvelD3, which, inversely, modulate claudin oligomerization
Cording, J., Berg, J., Käding, N., Bellmann, C., Tscheik, C., Westphal(*), J. K., Milatz(*), S., Günzel(*), D., Wolburg(*), H., Piontek, J., Huber(*), O.; Blasig, I. E.
J Cell Sci, 126:554-564

Tags: Molecular and Cell Physiology (Blasig, IE)

Abstract: Tight junctions seal the paracellular cleft of epithelia and endothelia, form vital barriers between tissue compartments and consist of tight-junction-associated marvel proteins (TAMPs) and claudins. The function of TAMPs and the interaction with claudins are not understood. We therefore investigated the binding between the TAMPs occludin, tricellulin, and marvelD3 and their interaction with claudins in living tight-junction-free human embryonic kidney-293 cells. In contrast to claudins and occludin, tricellulin and marvelD3 showed no enrichment at cell-cell contacts indicating lack of homophilic trans-interaction between two opposing cell membranes. However, occludin, marvelD3 and tricellulin exhibited homophilic cis-interactions, along one plasma membrane, as measured by fluorescence resonance energy transfer. MarvelD3 also cis-interacted with occludin and tricellulin heterophilically. Classic claudins, such as claudin-1 to -5 may show cis-oligomerization with TAMPs, whereas the non-classic claudin-11 did not. Claudin-1 and -5 improved enrichment of occludin and tricellulin at cell-cell contacts. The low mobile claudin-1 reduced the membrane mobility of the highly mobile occludin and tricellulin, as studied by fluorescence recovery after photobleaching. Co-transfection of claudin-1 with TAMPs led to changes of the tight junction strand network of this claudin to a more physiological morphology, depicted by freeze-fracture electron microscopy. The results demonstrate multilateral interactions between the tight junction proteins, in which claudins determine the function of TAMPs and vice versa, and provide deeper insights into the tight junction assembly.

A conformational intermediate in glutamate receptor activation
Lau(*), A. Y., Salazar, H., Blachowicz(*), L., Ghisi, V., Plested, A. J.; Roux(*), B.
Neuron, 79:492-503

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: Ionotropic glutamate receptors (iGluRs) transduce the chemical signal of neurotransmitter release into membrane depolarization at excitatory synapses in the brain. The opening of the transmembrane ion channel of these ligand-gated receptors is driven by conformational transitions that are induced by the association of glutamate molecules to the ligand-binding domains (LBDs). Here, we describe the crystal structure of a GluA2 LBD tetramer in a configuration that involves an approximately 30 degrees rotation of the LBD dimers relative to the crystal structure of the full-length receptor. The configuration is stabilized by an engineered disulfide crosslink. Biochemical and electrophysiological studies on full-length receptors incorporating either this crosslink or an engineered metal bridge show that this LBD configuration corresponds to an intermediate state of receptor activation. GluA2 activation therefore involves a combination of both intra-LBD (cleft closure) and inter-LBD dimer conformational transitions. Overall, these results provide a comprehensive structural characterization of an iGluR intermediate state.

Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate
Posor, Y., Eichhorn-Grünig, M., Puchkov, D., Schöneberg(*), J., Ullrich(*), A., Lampe, A., Müller(*), R., Zarbakhsh(*), S., Gulluni(*), F., Hirsch(*), E., Krauss, M., Schultz(*), C., Schmoranzer, J., Noe(*), F.; Haucke, V.
Nature, 499:233-+

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Phosphoinositides serve crucial roles in cell physiology, ranging from cell signalling to membrane traffic(1,2). Among the seven eukaryotic phosphoinositides the best studied species is phosphatidylinositol-4,5-bisphosphate (PI(4,5)P-2), which is concentrated at the plasma membrane where, among other functions, it is required for the nucleation of endocytic clathrin-coated pits(3-6). No phosphatidylinositol other than PI(4,5)P-2 has been implicated in clathrin-mediated endocytosis, whereas the subsequent endosomal stages of the endocytic pathway are dominated by phosphatidylinositol-3-phosphates(PI(3)P)(7). How phosphatidylinositol conversion from PI(4,5)P-2-positive endocytic intermediates to PI(3)P-containing endosomes is achieved is unclear. Here we show that formation of phosphatidylinositol-3,4-bisphosphate (PI(3,4)P-2) by class II phosphatidylinositol-3-kinase C2 alpha (PI(3) K C2 alpha) spatiotemporally controls clathrin-mediated endocytosis. Depletion of PI(3,4)P-2 or PI(3)K C2 alpha impairs the maturation of late-stage clathrin-coated pits before fission. Timed formation of PI(3,4)P-2 by PI(3)K C2 alpha is required for selective enrichment of the BAR domain protein SNX9 at late-stage endocytic intermediates. These findings provide a mechanistic framework for the role of PI(3,4)P-2 in endocytosis and unravel a novel discrete function of PI(3,4)P-2 in a central cell physiological process.

Combinatorial approach to drastically enhance the monoclonal antibody efficacy in targeted tumor therapy.
Gilabert-Oriol(*), R., Thakur(*), M., von Mallinckrodt(*), B., Hug(*), T., Wiesner, B., Eichhorst, J., Melzig(*), M. F., Fuchs(*), H.; Weng(*), A.
Mol Cancer Ther, 12

Tags: Cellular Imaging (Wiesner)

Modified Trastuzumab and Cetuximab Mediate Efficient Toxin Delivery While Retaining Antibody-Dependent Cell-Mediated Cytotoxicity in Target Cells
Gilabert-Oriol(*), R., Thakur(*), M., von Mallinckrodt(*), B., Hug(*), T., Wiesner, B., Eichhorst, J., Melzig(*), M. F., Fuchs(*), H.; Weng(*), A.
Mol Pharmaceut, 10:4347-4357

Tags: Cellular Imaging (Wiesner)

Abstract: Monoclonal antibody-based therapy is one of the most successful strategies for treatment of cancer. However, the insufficient cell killing activity of monoclonal antibodies limits their therapeutic potential. These limitations can be overcome by the application of immunotoxins, which consist of a monoclonal antibody that specifically delivers a toxin into the cancer cell. An ideal immunotoxin combines the functionality of the monoclonal antibody (antagonistic binding to targeted receptors and interaction with the innate immune system) with the cell-killing activity of the toxic moiety. In addition, it should be sensitive for certain triterpenoid saponins that are known to lead to a tremendous augmentation of the antitumoral efficacy of the immunotoxin. In this study, the monoclonal antibodies trastuzumab (Herceptin) and cetuximab (Erbitux) were conjugated via cleavable disulfide bonds to the plant derived toxin saporin. The ability of the modified tumor-specific therapeutic antibodies to deliver their toxic payload into the target cells was investigated by impedance-based real-time viability assays and confocal live cell imaging. We further provide evidence that the immunotoxins retained their ability to trigger antibody-dependent cell-mediated cytotoxicity. They specifically bound to their target cell receptor, and their cell-killing activity was drastically augmented in the presence of triterpenoid saponins. Further mechanistic studies indicated a specific saponin-mediated endo/lysosomal release of the toxin moiety. These results open a promising avenue to overcome the present limitations of therapeutic antibodies and to achieve a higher antitumoral efficacy in cancer therapy.

Macromolecular interactions of triterpenoids and targeted toxins: Role of saponins charge
Thakur(*), M., Weng(*), A., Pieper(*), A., Mergel(*), K., von Mallinckrodt(*), B., Gilabert-Oriol(*), R., Gorick(*), C., Wiesner, B., Eichhorst, J., Melzig(*), M. F.; Fuchs(*), H.
Int J Biol Macromol, 61:285-294

Tags: Cellular Imaging (Wiesner)

Abstract: Macromolecular interaction of protein toxins with certain plant triterpenoids holds potential for application in tumor therapy. The ability of only certain saponins to enhance the endosomal escape of toxins specifically in tumor cells was evaluated and set into correlation with the electrophoretic mobility. Saponins from Saponaria officinalis Linn, were selected as a lead to understand this evolutionarily conserved principle in detail. Agarose gel electrophoresis was utilized to procure pure saponin fractions with different electrophoretic mobility, which were tested for their ability to enhance the toxicity by live cell monitoring. Five fractions (SOG1-SOG5) were isolated with a relative electrophoretic mobility of (-0.05, 0.41, 0.59, 0.75 and 1.00) and evaluated using thin layer chromatography, HPLC, and mass spectroscopic analysis. Cytotoxicity experiments revealed highest effectiveness with SOG3. Live cell imaging experiments with SOG3 revealed that this saponin with a specific REM of 0.59 could assist in the lyso/endosomal release of the toxic payload without affecting the integrity of plasma membrane and could lead to the induction of apoptosis. This charge dependent enhancement was also found to be highly specific to type I ribosome inactivating proteins compared to bacterial toxins. Charge interaction of plant toxins and saponins with tumor cells, plays a major role in toxin specific modulation of response. The finding opens up newer ways of finding protein saponin interaction conserved evolutionarily and to test their role in endosomal escape of therapeutic molecules. (C) 2013 Elsevier B.V. All rights reserved.

Dynamic nuclear polarization of spherical nanoparticles
Akbey, Ü., Altin(*), B., Linden, A., Ozcelik(*), S., Gradzielski(*), M.; Oschkinat, H.
Phys Chem Chem Phys, 15:20706-20716

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Spherical silica nanoparticles of various particle sizes (~10 to 100 nm), produced by a modified Stoeber method employing amino acids as catalysts, are investigated using Dynamic Nuclear Polarization (DNP) enhanced Nuclear Magnetic Resonance (NMR) spectroscopy. This study includes ultra-sensitive detection of surface-bound amino acids and their supramolecular organization in trace amounts, exploiting the increase in NMR sensitivity of up to three orders of magnitude via DNP. Moreover, the nature of the silicon nuclei on the surface and the bulk silicon nuclei in the core (sub-surface) is characterized at atomic resolution. Thereby, we obtain unique insights into the surface chemistry of these nanoparticles, which might result in improving their rational design as required for promising applications, e.g. as catalysts or imaging contrast agents. The non-covalent binding of amino acids to surfaces was determined which shows that the amino acids not just function as catalysts but become incorporated into the nanoparticles during the formation process. As a result only three distinct Q-types of silica signals were observed from surface and core regions. We observed dramatic changes of DNP enhancements as a function of particle size, and very small particles (which suit in vivo applications better) were hyperpolarized with the best efficiency. Nearly one order of magnitude larger DNP enhancement was observed for nanoparticles with 13 nm size compared to particles with 100 nm size. We determined an approximate DNP penetration-depth (~4.2 or ~5.7 nm) for the polarization transfer from electrons to the nuclei of the spherical nanoparticles. Faster DNP polarization buildup was observed for larger nanoparticles. Efficient hyperpolarization of such nanoparticles, as achieved in this work, can be utilized in applications such as magnetic resonance imaging (MRI).

Human mast cell line-1 (HMC-1) cells exhibit a membrane capacitance increase when dialysed with high free-Ca(2+) and GTPgammaS containing intracellular solution
Balletta(*), A., Lorenz, D., Rummel(*), A., Gerhard(*), R., Bigalke(*), H.; Wegner(*), F.
Eur J Pharmacol, 720:227-236

Tags: Cellular Imaging (Wiesner)

Abstract: An increase in cytosolic free calcium concentration [Ca(2+)]i initiates the exocytotic activity in various types of secretory cells. The guanosine 5'-O-[3-thio]triphosphate (GTPgammaS), a non-hydrolysable analogue of GTP (guanosine 5'-triphosphate), is an effective secretagogue for different cell types of different species, like mast cells, neutrophils or eosinophils. Consequently, the internal administration of GTPgammaS causes degranulation of mouse and rat mast cells. Regarding rat mast cells, it is proved that Ca(2+) can cooperate with GTP or GTPgammaS in accelerating and increasing amplitude of the secretory response. All the previous studies with respect to capacitance recordings and mast cells were performed using mouse or rat mast cells, usually derived from peritoneum or the rat basophilic leukaemia cell line RBL. In this study, we applied the capacitance measurement technique to the human mast cell line-1 (HMC-1) cells, an immature cell line established from a patient with mast cell leukaemia. Patch-clamp dialysis experiments revealed that high intracellular free Ca(2+) and GTPgammaS concentrations are both required for considerable capacitance increases in HMC-1 cells. During degranulation of HMC-1 cells, the total membrane capacitance (Cm) increase appeared continuously and, in some cases, as a discrete capacitance change, developing in a stepwise manner. Then, we tested the effect of latrunculin B upon HMC-1 cell capacitance increase as well as of some classic mast cell stimulators like PMA, A23187 and IL-1beta in hexosaminidase release. Finally, we could conclude that the HMC-1 cell line represents a suitable model for the study of human mast cell degranulation.

Potentiation of the Transient Receptor Potential Vanilloid 1 Channel Contributes to Pruritogenesis in a Rat Model of Liver Disease
Belghiti(*), M., Estevez-Herrera(*), J., Gimenez-Garzo(*), C., Gonzalez-Usano(*), A., Montoliu(*), C., Ferrer-Montiel(*), A., Felipo(*), V.; Planells-Cases, R.
Journal of Biological Chemistry, 288:9675-9685

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Persistent pruritus is a common disabling dermatologic symptom associated with different etiologic factors. These include primary skin conditions, as well as neuropathic, psychogenic, or systemic disorders like chronic liver disease. Defective clearance of potential pruritogenic substances that activate itch-specific neurons innervating the skin is thought to contribute to cholestatic pruritus. However, because the underlying disease-specific pruritogens and itch-specific neuronal pathways and mechanism(s) are unknown, symptomatic therapeutic intervention often leads to no or only limited success. In the current study, we aimed to first validate rats with bile duct ligation (BDL) as a model for hepatic pruritus and then to evaluate the contribution of inflammation, peripheral neuronal sensitization, and specific signaling pathways and subpopulations of itch-responsive neurons to scratching behavior and thermal hypersensitivity. Chronic BDL rats displayed enhanced scratching behavior and thermal hyperalgesia indicative of peripheral neuroinflammation. BDL-induced itch and hypersensitivity involved a minor contribution of histaminergic/serotonergic receptors, but significant activation of protein-activated receptor 2 (PAR(2)) receptors, prostaglandin PGE(2) formation, and potentiation of transient receptor potential vanilloid 1 (TRPV1) channel activity. The sensitization of dorsal root ganglion nociceptors in BDL rats was associated with increased surface expression of PAR(2) and TRPV1 proteins and an increase in the number of PAR(2)-and TRPV1-expressing peptidergic neurons together with a shift of TRPV1 receptor expression to medium sized dorsal root ganglion neurons. These results suggest that pruritus and hyperalgesia in chronic cholestatic BDL rats are associated with neuroinflammation and involve PAR(2)-induced TRPV1 sensitization. Thus, pharmacological modulation of PAR2 and/or TRPV1 may be a valuable therapeutic approach for patients with chronic liver pruritus refractory to conventional treatments.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)

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