FMP Publications

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

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Hydrogen bonding involving side chain exchangeable groups stabilizes amyloid quarternary structure
Agarwal, V., Linser, R., Dasari, M., Fink, U., del Amo, J. M.; Reif, B.
Phys Chem Chem Phys, 15:12551-12557

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: The amyloid beta-peptide (Abeta) is the major structural component of amyloid fibrils in the plaques of brains of Alzheimer's disease patients. Numerous studies have addressed important aspects of secondary and tertiary structure of fibrils. In electron microscopic images, fibrils often bundle together. The mechanisms which drive the association of protofilaments into bundles of fibrils are not known. We show here that amino acid side chain exchangeable groups like e.g. histidines can provide useful restraints to determine the quarternary assembly of an amyloid fibril. Exchangeable protons are only observable if a side chain hydrogen bond is formed and the respective protons are protected from exchange. The method relies on deuteration of the Abeta peptide. Exchangeable deuterons are substituted with protons, before fibril formation is initiated.

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.

NMR spectroscopy reveals unexpected structural variation at the protein-protein interface in MHC class I molecules
Beerbaum, M., Ballaschk, M., Erdmann, N., Schnick(*), C., Diehl, A., Uchanska-Ziegler(*), B., Ziegler(*), A.; Schmieder, P.
J Biomol NMR, 57:167-178

Tags: Solution NMR (Schmieder)

Abstract: beta2-Microglobulin (beta2m) is a small, monomorphic protein non-covalently bound to the heavy chain (HC) in polymorphic major histocompatibility complex (MHC) class I molecules. Given the high evolutionary conservation of structural features of beta2m in various MHC molecules as shown by X-ray crystallography, beta2m is often considered as a mere scaffolding protein. Using nuclear magnetic resonance (NMR) spectroscopy, we investigate here whether beta2m residues at the interface to the HC exhibit changes depending on HC polymorphisms and the peptides bound to the complex in solution. First we show that human beta2m can effectively be produced in deuterated form using high-cell-density-fermentation and we employ the NMR resonance assignments obtained for triple-labeled beta2m bound to the HLA-B*27:09 HC to examine the beta2m-HC interface. We then proceed to compare the resonances of beta2m in two minimally distinct subtypes, HLA-B*27:09 and HLA-B*27:05, that are differentially associated with the spondyloarthropathy Ankylosing Spondylitis. Each of these subtypes is complexed with four distinct peptides for which structural information is already available. We find that only the resonances at the beta2m-HC interface show a variation of their chemical shifts between the different complexes. This indicates the existence of an unexpected plasticity that enables beta2m to accommodate changes that depend on HC polymorphism as well as on the bound peptide through subtle structural variations of the protein-protein interface.

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.

A Supramolecular Peptide Synthesizer
Bertran-Vicente, J.; Hackenberger, C. P. R.
Angew Chem Int Edit, 52:6140-6142

Tags: Chemical Biology II (Hackenberger)

The new KCNQ2 activator 4-Chlor-N-(6-chlor-pyridin-3-yl)-benzamid displays anticonvulsant potential
Boehlen(*), A., Schwake(*), M., Dost(*), R., Kunert(*), A., Fidzinski, P., Heinemann(*), U.; Gebhardt(*), C.
Brit J Pharmacol, 168:1182-1200

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Background and Purpose KCNQ2-5 channels are voltage-gated potassium channels that regulate neuronal excitability and represent suitable targets for the treatment of hyperexcitability disorders. The effect of Chlor-N-(6-chlor-pyridin-3-yl)-benzamid was tested on KCNQ subtypes for its ability to alter neuronal excitability and for its anticonvulsant potential. Experimental Approach The effect of 4-Chlor-N-(6-chlor-pyridin-3-yl)-benzamid was evaluated using whole-cell voltage-clamp recordings from CHO cells and Xenopus laevis oocytes expressing different types of KCNQ channels. Epileptiform afterdischarges were recorded in fully amygdala-kindled rats in vivo. Neuronal excitability was assessed using field potential and whole cell recording in rat hippocampus in vitro. Key Results 4-Chlor-N-(6-chlor-pyridin-3-yl)-benzamid caused a hyperpolarizing shift of the activation curve and a pronounced slowing of deactivation in KCNQ2-mediated currents, whereas KCNQ3/5 heteromers remained unaffected. The effect was also apparent in the Retigabine-insensitive mutant KCNQ2-W236L. In fully amygdala-kindled rats, it elevated the threshold for induction of afterdischarges and reduced seizure severity and duration. In hippocampal CA1 cells, 4-Chlor-N-(6-chlor-pyridin-3-yl)-benzamid strongly damped neuronal excitability caused by a membrane hyperpolarization and a decrease in membrane resistance and induced an increase of the somatic resonance frequency on the single cell level, whereas synaptic transmission was unaffected. On the network level, 4-Chlor-N-(6-chlor-pyridin-3-yl)-benzamid caused a significant reduction of and oscillation peak power, with no significant change in oscillation frequency. Conclusion and Implications Our data indicate that 4-Chlor-N-(6-chlor-pyridin-3-yl)-benzamid is a potent KCNQ activator with a selectivity for KCNQ2 containing channels. It strongly reduces neuronal excitability and displays anticonvulsant activity in vivo.

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.

A Well-Defined Pd Hybrid Material for the Z-Selective Semihydrogenation of Alkynes Characterized at the Molecular Level by DNP SENS
Conley(*), M. P., Drost(*), R. M., Baffert(*), M., Gajan(*), D., Elsevier(*), C., Franks, W. T., Oschkinat, H., Veyre(*), L., Zagdoun(*), A., Rossini(*), A., Lelli(*), M., Lesage(*), A., Casano(*), G., Ouari(*), O., Tordo(*), P., Emsley(*), L., Coperet(*), C.; Thieuleux(*), C.
Chem-Eur J, 19:12234-12238

Tags: NMR-Supported Structural Biology (Oschkinat)

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
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13125 Berlin, Germany
+4930 94793 - 100 
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