FMP Publications

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

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References per page: Show keywords Show abstracts
Lipid-Labeling Facilitates a Novel Magnetic Isolation Procedure to Characterize Pathogen-Containing Phagosomes
Steinhäuser(*), C., Heigl(*), U., Tchikov(*), V., Schwarz(*), J., Gutsmann(*), T., Seeger(*), K., Brandenburg(*), J., Fritsch(*), J., Schroeder(*), J., Wiesmüller(*), K. H., Rosenkrands(*), I., Walther(*), P., Pott(*), J., Krause, E., Ehlers(*), S., Schneider-Brachert(*), W., Schütze(*), S.; Reiling(*), N.
Traffic, 14:321-336

Tags: Mass Spectrometry (Krause, E.)

Abstract: Here we describe a novel approach for the isolation and biochemical characterization of pathogen-containing compartments from primary cells: We developed a lipid-based procedure to magnetically label the surface of bacteria and visualized the label by scanning and transmission electron microscopy (SEM, TEM). We performed infection experiments with magnetically labeled Mycobacterium avium, M. tuberculosis and Listeria monocytogenes and isolated magnetic bacteria-containing phagosomes using a strong magnetic field in a novel free-flow system. Magnetic labeling of M.tuberculosis did not affect the virulence characteristics of the bacteria during infection experiments addressing host cell activation, phagosome maturation delay and replication in macrophages in vitro. Biochemical analyses of the magnetic phagosome-containing fractions provided evidence of an enhanced presence of bacterial antigens and a differential distribution of proteins involved in the endocytic pathway over time as well as cytokine-dependent changes in the phagosomal protein composition. The newly developed method represents a useful approach to characterize and compare pathogen-containing compartments, in order to identify microbial and host cell targets for novel anti-infective strategies.

Small-molecule screening identifies modulators of aquaporin-2 trafficking
Bogum, J., Faust(*), D., Zühlke, K., Eichhorst, J., Moutty, M. C., Furkert, J., Eldahshan(*), A., Neuenschwander, M., von Kries, J. P., Wiesner, B., Trimpert(*), C., Deen(*), P. M., Valenti(*), G., Rosenthal(*), W.; Klussmann(*), E.
Journal of the American Society of Nephrology : JASN, 24:744-758

Tags: Cellular Imaging (Wiesner), Screening Unit (von Kries), Anchored Signaling (Klussmann)

Abstract: In the principal cells of the renal collecting duct, arginine vasopressin (AVP) stimulates the synthesis of cAMP, leading to signaling events that culminate in the phosphorylation of aquaporin-2 water channels and their redistribution from intracellular domains to the plasma membrane via vesicular trafficking. The molecular mechanisms that control aquaporin-2 trafficking and the consequent water reabsorption, however, are not completely understood. Here, we used a cell-based assay and automated immunofluorescence microscopy to screen 17,700 small molecules for inhibitors of the cAMP-dependent redistribution of aquaporin-2. This approach identified 17 inhibitors, including 4-acetyldiphyllin, a selective blocker of vacuolar H(+)-ATPase that increases the pH of intracellular vesicles and causes accumulation of aquaporin-2 in the Golgi compartment. Although 4-acetyldiphyllin did not inhibit forskolin-induced increases in cAMP formation and downstream activation of protein kinase A (PKA), it did prevent cAMP/PKA-dependent phosphorylation at serine 256 of aquaporin-2, which triggers the redistribution to the plasma membrane. It did not, however, prevent cAMP-induced changes to the phosphorylation status at serines 261 or 269. Last, we identified the fungicide fluconazole as an inhibitor of cAMP-mediated redistribution of aquaporin-2, but its target in this pathway remains unknown. In conclusion, our screening approach provides a method to begin dissecting molecular mechanisms underlying AVP-mediated water reabsorption, evidenced by our identification of 4-acetyldiphyllin as a modulator of aquaporin-2 trafficking.

Culturing Primary Rat Inner Medullary Collecting Duct Cells
Faust(*), D., Geelhaar(*), A., Eisermann(*), B., Eichhorst, J., Wiesner, B., Rosenthal(*), W.; Klussmann(*), E.
Jove-J Vis Exp,

Tags: Cellular Imaging (Wiesner)

Abstract: Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion. Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

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.

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)

Structural insights into the mechanism of GTPase activation in the GIMAP family
Schwefel(*), D., Arasu(*), B. S., Marino(*), S. F., Lamprecht(*), B., Kochert(*), K., Rosenbaum(*), E., Eichhorst, J., Wiesner, B., Behlke(*), J., Rocks(*), O., Mathas(*), S.; Daumke(*), O.
Structure (London, England : 1993), 21:550-559

Tags: Cellular Imaging (Wiesner)

Abstract: GTPases of immunity-associated proteins (GIMAPs) are regulators of lymphocyte survival and homeostasis. We previously determined the structural basis of GTP-dependent GIMAP2 scaffold formation on lipid droplets. To understand how its GTP hydrolysis is activated, we screened for other GIMAPs on lipid droplets and identified GIMAP7. In contrast to GIMAP2, GIMAP7 displayed dimerization-stimulated GTP hydrolysis. The crystal structure of GTP-bound GIMAP7 showed a homodimer that assembled via the G domains, with the helical extensions protruding in opposite directions. We identified a catalytic arginine that is supplied to the opposing monomer to stimulate GTP hydrolysis. GIMAP7 also stimulated GTP hydrolysis by GIMAP2 via an analogous mechanism. Finally, we found GIMAP2 and GIMAP7 expression differentially regulated in several human T cell lymphoma lines. Our findings suggest that GTPase activity in the GIMAP family is controlled by homo- and heterodimerization. This may have implications for the differential roles of some GIMAPs in lymphocyte survival.

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.

Exome sequencing reveals new causal mutations in children with epileptic encephalopathies
Veeramah(*), K. R., Johnstone(*), L., Karafet(*), T. M., Wolf(*), D., Sprissler(*), R., Salogiannis(*), J., Barth-Maron(*), A., Greenberg(*), M. E., Stuhlmann, T., Weinert, S., Jentsch, T. J., Pazzi(*), M., Restifo(*), L. L., Talwar(*), D., Erickson(*), R. P.; Hammer(*), M. F.
Epilepsia, 54:1270-1281

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Purpose: The management of epilepsy in children is particularly challenging when seizures are resistant to antiepileptic medications, or undergo many changes in seizure type over time, or have comorbid cognitive, behavioral, or motor deficits. Despite efforts to classify such epilepsies based on clinical and electroencephalographic criteria, many children never receive a definitive etiologic diagnosis. Whole exome sequencing (WES) is proving to be a highly effective method for identifying de novo variants that cause neurologic disorders, especially those associated with abnormal brain development. Herein we explore the utility of WES for identifying candidate causal de novo variants in a cohort of children with heterogeneous sporadic epilepsies without etiologic diagnoses. Methods: We performed WES (mean coverage approximately 403) on 10 trios comprised of unaffected parents and a child with sporadic epilepsy characterized by difficult-to-control seizures and some combination of developmental delay, epileptic encephalopathy, autistic features, cognitive impairment, or motor deficits. Sequence processing and variant calling were performed using standard bioinformatics tools. A custom filtering system was used to prioritize de novo variants of possible functional significance for validation by Sanger sequencing. Key Findings: In 9 of 10 probands, we identified one or more de novo variants predicted to alter protein function, for a total of 15. Four probands had de novo mutations in genes previously shown to harbor heterozygous mutations in patients with severe, early onset epilepsies (two in SCN1A, and one each in CDKL5 and EEF1A2). In three children, the de novo variants were in genes with functional roles that are plausibly relevant to epilepsy (KCNH5, CLCN4, and ARHGEF15). The variant in KCNH5 alters one of the highly conserved arginine residues of the voltage sensor of the encoded voltage-gated potassium channel. In vitro analyses using cell-based assays revealed that the CLCN4 mutation greatly impaired ion transport by the ClC-4 2Cl(-)/H+-exchanger and that the mutation in ARHGEF15 reduced GEF exchange activity of the gene product, Ephexin5, by about 50%. Of interest, these seven probands all presented with seizures within the first 6 months of life, and six of these have intractable seizures. Significance: The finding that 7 of 10 children carried de novo mutations in genes of known or plausible clinical significance to neuronal excitability suggests that WES will be of use for the molecular genetic diagnosis of sporadic epilepsies in children, especially when seizures are of early onset and difficult to control.

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.

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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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