FMP Publications

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

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References
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
(2013)

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.

Highly functionalized terpyridines as competitive inhibitors of AKAP-PKA interactions
Schäfer(*), G., Milic(*), J., Eldahshan, A., Götz(*), F., Zühlke(*), K., Schillinger, C., Kreuchwig, A., Elkins(*), J. M., Abdul Azeez(*), K. R., Oder(*), A., Moutty(*), M. C., Masada(*), N., Beerbaum, M., Schlegel, B., Niquet(*), S., Schmieder, P., Krause, G., von Kries, J. P., Cooper(*), D. M., Knapp(*), S., Rademann, J., Rosenthal(*), W.; Klussmann(*), E.
Angew Chem Int Ed Engl, 52:12187-12191
(2013)

Tags: Medicinal Chemistry (Rademann), Screening Unit (von Kries), Solution NMR (Schmieder)

Mapping discontinuous protein-binding sites via structure-based peptide libraries: combining in silico and in vitro approaches
Jaeger(*), I. S., Kretzschmar(*), I., Körner, J., Weiser(*), A. A., Mahrenholz(*), C. C., Potty(*), A., Kourentzi(*), K., Willson(*), R. C., Volkmer(*), R.; Preissner(*), R.
J Mol Recognit, 26:23-31
(2013)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: To perform their various functions, protein surfaces often have to interact with each other in a specific way. Usually, only parts of a protein are accessible and can act as binding sites. Because proteins consist of polypeptide chains that fold into complex three-dimensional shapes, binding sites can be divided into two different types: linear sites that follow the primary amino acid sequence and discontinuous binding sites, which are made up of short peptide fragments that are adjacent in spatial proximity. Such discontinuous binding sites dominate proteinprotein interactions, but are difficult to identify. To meet this challenge, we combined a computational, structure-based approach and an experimental, high-throughput method. SUPERFICIAL is a program that uses protein structures as input and generates peptide libraries to represent the protein's surface. A large number of the predicted peptides can be simultaneously synthesised applying the SPOT technology. The results of a binding assay subsequently help to elucidate proteinprotein interactions; the approach is applicable to any kind of protein. The crystal structure of the complex of hen egg lysozyme with the well-characterised murine IgG1 antibody HyHEL-5 is available, and the complex is known to have a discontinuous binding site. Using SUPERFICIAL, the entire surface of lysozyme was translated into a peptide library that was synthesised on a cellulose membrane using the SPOT technology and tested against the HyHEL-5 antibody. In this way, it was possible to identify two peptides (longest common sequence and peptide 19) that represented the discontinuous epitope of lysozyme. Copyright (c) 2012 John Wiley & Sons, Ltd.

A Floquet description of phase alternated sequences for efficient homonuclear recoupling in solid perdeuterated systems
Jayanthi(*), S., Akbey, Ü., Uluca(*), B., Oschkinat, H.; Vega(*), S.
Journal of Magnetic Resonance, 234:10-20
(2013)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: A Floquet description of a phase alternated homonuclear recoupling scheme for perdeuterated systems is presented. As a result, we demonstrate improvements in the recoupling efficiency of the DOuble Nucleus Enhanced Recoupling [DONER; J. Am. Chem. Soc. 131 (2009) 170541 technique by utilizing Phase Alternated Recoupling Irradiation Schemes [PARIS; Chem. Phys. Lett. 469 (2009) 342]. The effect of proton and deuterium radio frequency irradiation during recoupling has been systematically studied and theoretical observations have been verified experimentally using a deuterated model compound, L-Alanine, at 10 and 20 kHz magic angle spinning frequency. Experimental results are well in agreement with theoretical observations, thereby significantly increasing the recoupling efficiency of conventional DONER in perdeuterated systems. (C) 2013 Elsevier Inc. All rights reserved.

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
(2013)

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
(2013)

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
(2013)

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.

The mechanism of denaturation and the unfolded state of the alpha-helical membrane-associated protein Mistic
Jacso, T., Bardiaux, B., Broecker(*), J., Fiedler(*), S., Baerwinkel(*), T., Mainz, A., Fink, U., Vargas(*), C., Oschkinat, H., Keller(*), S.; Reif, B.
J Am Chem Soc, 135:18884-18891
(2013)

Tags: Solid-State NMR Spectroscopy (Reif), NMR-Supported Structure Biology (Oschkinat)

Abstract: In vitro protein-folding studies using chemical denaturants such as urea are indispensible in elucidating the forces and mechanisms determining the stability, structure, and dynamics of water-soluble proteins. By contrast, alpha-helical membrane-associated proteins largely evade such approaches because they are resilient to extensive unfolding. We have used optical and NMR spectroscopy to provide an atomistic-level dissection of the effects of urea on the structure and dynamics of the alpha-helical membrane-associated protein Mistic as well as its interactions with detergent and solvent molecules. In the presence of the zwitterionic detergent lauryl dimethylamine oxide, increasing concentrations of urea result in a complex sequence of conformational changes that go beyond simple two-state unfolding. Exploiting this finding, we report the first high-resolution structural models of the urea denaturation process of an alpha-helical membrane-associated protein and its completely unfolded state, which contains almost no regular secondary structure but nevertheless retains a topology close to that of the folded state.

CRIS-A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending
Krähling(*), A. M., Alvarez(*), L., Debowski(*), K., Van(*), Q., Gunkel(*), M., Irsen(*), S., Al-Amoudi(*), A., Strünker(*), T., Kremmer(*), E., Krause, E., Voigt(*), I., Wortge(*), S., Waisman(*), A., Weyand(*), I., Seifert(*), R., Kaupp(*), U. B.; Wachten(*), D.
Plos Genet, 9
(2013)

Tags: Mass Spectrometry (Krause, E.)

Abstract: The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD). Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function) that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca2+ regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca2+-regulated proteins that-in mature sperm-are involved in flagellar bending.

Signal Peptide Cleavage from GP5 of PRRSV: A Minor Fraction of Molecules Retains the Decoy Epitope, a Presumed Molecular Cause for Viral Persistence
Thaa(*), B., Sinhadri(*), B. C., Tielesch(*), C., Krause, E.; Veit(*), M.
Plos One, 8
(2013)

Tags: Mass Spectrometry (Krause, E.)

Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) is the major pathogen in the pig industry. Variability of the antigens and persistence are the biggest challenges for successful control and elimination of the disease. GP5, the major glycoprotein of PRRSV, is considered an important target of neutralizing antibodies, which however appear only late in infection. This was attributed to the presence of a "decoy epitope" located near a hypervariable region of GP5. This region also harbors the predicted signal peptide cleavage sites and (dependent on the virus strain) a variable number of potential N-glycosylation sites. Molecular processing of GP5 has not been addressed experimentally so far: whether and where the signal peptide is cleaved and (as a consequence) whether the "decoy epitope" is present in virus particles. We show that the signal peptide of GP5 from the American type 2 reference strain VR-2332 is cleaved, both during in vitro translation in the presence of microsomes and in transfected cells. This was found to be independent of neighboring glycosylation sites and occurred in a variety of porcine cells for GP5 sequences derived from various type 2 strains. The exact signal peptide cleavage site was elucidated by mass spectrometry of virus-derived and recombinant GP5. The results revealed that the signal peptide of GP5 is cleaved at two sites. As a result, a mixture of GP5 proteins exists in virus particles, some of which still contain the "decoy epitope" sequence. Heterogeneity was also observed for the use of glycosylation sites in the hypervariable region. Lastly, GP5 mutants were engineered where one of the signal peptide cleavage sites was blocked. Wildtype GP5 exhibited exactly the same SDS-PAGE mobility as the mutant that is cleavable at site 2 only. This indicates that the overwhelming majority of all GP5 molecules does not contain the "decoy epitope".

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)
info(at)fmp-berlin.de

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