FMP Publications

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

Year:  
All :: 2010, ... , 2012, 2013, 2014, ... , 2017
Author:  
All :: (, A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y, Z 
All :: Ma(*), ... , Merk(*), Merlo(*), Mertins(*), ... , Mylona(*) 
Preferences: 
References per page: Show keywords Show abstracts
References
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.

Inhibition of clathrin by pitstop 2 activates the spindle assembly checkpoint and induces cell death in dividing HeLa cancer cells
Smith(*), C. M., Haucke, V., McCluskey(*), A., Robinson(*), P. J.; Chircop(*), M.
Mol Cancer, 12
(2013)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Background: During metaphase clathrin stabilises the mitotic spindle kinetochore(K)-fibres. Many anti-mitotic compounds target microtubule dynamics. Pitstop 2 (TM) is the first small molecule inhibitor of clathrin terminal domain and inhibits clathrin-mediated endocytosis. We investigated its effects on a second function for clathrin in mitosis. Results: Pitstop 2 did not impair clathrin recruitment to the spindle but disrupted its function once stationed there. Pitstop 2 trapped HeLa cells in metaphase through loss of mitotic spindle integrity and activation of the spindle assembly checkpoint, phenocopying clathrin depletion and aurora A kinase inhibition. Conclusions: Pitstop 2 is therefore a new tool for investigating clathrin spindle dynamics. Pitstop 2 reduced viability in dividing HeLa cells, without affecting dividing non-cancerous NIH3T3 cells, suggesting that clathrin is a possible novel anti-mitotic drug target.

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.

Compromised fidelity of endocytic synaptic vesicle protein sorting in the absence of stonin 2
Kononenko, N. L., Diril(*), M. K., Puchkov, D., Kintscher(*), M., Koo(*), S. J., Pfuhl(*), G., Winter(*), Y., Wienisch(*), M., Klingauf(*), J., Breustedt(*), J., Schmitz(*), D., Maritzen, T.; Haucke, V.
Proc Natl Acad Sci U S A, 110:E526-535
(2013)

Tags: Molecular Pharmacology and Cell Biology (Haucke),Membrane Traffic and Cell Motility (Maritzen)

Abstract: Neurotransmission depends on the exocytic fusion of synaptic vesicles (SVs) and their subsequent reformation either by clathrin-mediated endocytosis or budding from bulk endosomes. How synapses are able to rapidly recycle SVs to maintain SV pool size, yet preserve their compositional identity, is poorly understood. We demonstrate that deletion of the endocytic adaptor stonin 2 (Stn2) in mice compromises the fidelity of SV protein sorting, whereas the apparent speed of SV retrieval is increased. Loss of Stn2 leads to selective missorting of synaptotagmin 1 to the neuronal surface, an elevated SV pool size, and accelerated SV protein endocytosis. The latter phenotype is mimicked by overexpression of endocytosis-defective variants of synaptotagmin 1. Increased speed of SV protein retrieval in the absence of Stn2 correlates with an up-regulation of SV reformation from bulk endosomes. Our results are consistent with a model whereby Stn2 is required to preserve SV protein composition but is dispensable for maintaining the speed of SV recycling.

Traceless Staudinger acetylation of azides in aqueous buffers
Sowa(*), S., Mühlberg, M., Pietrusiewicz(*), K. M.; Hackenberger, C. P. R.
Bioorgan Med Chem, 21:3465-3472
(2013)

Tags: Chemical Biology II (Hackenberger)

Abstract: In this paper, we demonstrate the applicability of water-soluble p-dimethylaminoethyl substituted phosphinomethanethiol in acetyl transfer reactions by the traceless Staudinger ligation with unprotected e-azido lysine containing peptides in aqueous buffer systems. Additionally, we present an improved synthesis pathway for the water-soluble phosphinothiol linkers requiring less steps in a comparable overall yield in comparison to previously published protocols. (C) 2013 Elsevier Ltd. 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.

NMR Spectroscopy of Soluble Protein Complexes at One Mega-Dalton and Beyond
Mainz, A., Religa, T. L., Sprangers, R., Linser, R., Kay, L. E.; Reif, B.
Angew Chem Int Edit, 52:8746-8751
(2013)

Tags: Solid-State NMR Spectroscopy (Reif)

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.

Page:  
Previous | 1, 2, 3 | Next
Export as:
BibTeX, XML

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)
info(at)fmp-berlin.de

Like many sites, we use cookies to optimize the user's browsing experience. Data Protection OK