FMP Publications

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

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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.

Functional properties of cell-free expressed human endothelin A and endothelin B receptors in artificial membrane environments
Proverbio(*), D., Roos(*), C., Beyermann, M., Orban(*), E., Dötsch(*), V.; Bernhard(*), F.
Bba-Biomembranes, 1828:2182-2192

Tags: Peptide Chemistry (Beyermann)

Abstract: The human endothelin receptors are members of the rhodopsin class A of G-protein coupled receptors and key modulators of blood pressure regulation. Their functional in vitro characterization has widely been limited by the availability of high quality samples. We have optimized cell-free expression protocols for the human endothelin A and endothelin B receptors by implementing co-translational association approaches of the synthesized proteins with supplied liposomes or nanodiscs. Efficiency of membrane association and ligand binding properties of the receptors have systematically been studied in correlation to different membrane environments and lipid types. Ligand binding was analyzed by a number of complementary assays including radioassays, surface plasmon resonance and fluorescence measurements. High affinity binding of the peptide ligand ET-I to both endothelin receptors could be obtained with several conditions and the highest Bmax values were measured In association with nanodiscs. We could further obtain the characteristic differential binding pattern of the two endothelin receptors with a panel of selected agonists and antagonists. Two intrinsic properties of the functionally folded endothelin B receptor, the proteolytic processing based on conformational recognition as well as the formation of SDS-resistant complexes with the peptide ligand ET-1, were observed with samples obtained from several cell-free expression conditions. High affinity and specific binding of ligands could furthermore be obtained with non-purified receptor samples in crude cell-free reaction mixtures, thus providing new perspectives for fast in vitro screening applications. (C) 2013 Elsevier B.V. All rights reserved.

Greasing the synaptic vesicle cycle by membrane lipids
Puchkov, D.; Haucke, V.
Trends Cell Biol, 23:493-503

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Neurotransmission is based on the exocytic release of neurotransmitters from synaptic vesicles (SVs) at nerve terminals and the subsequent retrieval of SV membranes. Evidence from genetic analysis of model organisms, high-resolution imaging, and biochemical studies indicate that, in addition to the well-studied function of exo-endocytic protein networks, membrane lipids and their derivatives play a key role in SV cycling. These include structural lipids such as cholesterol and sphingolipids as well as phosphoinositides (PIs), which interact with select components of the exocytic and endocytic machineries, thereby coupling both limbs of the SV cycle. Here we provide an overview of the function of lipids in SV cycling and discuss potential models of how lipids and lipid-protein interactions may regulate presynaptic function.

Biological and Tumor-Promoting Effects of Dioxin-like and Non-Dioxin-like Polychlorinated Biphenyls in Mouse Liver After Single or Combined Treatment
Rignall(*), B., Grote(*), K., Gavrilov(*), A., Weimer(*), M., Kopp-Schneider(*), A., Krause, E., Appel(*), K. E., Buchmann(*), A., Robertson(*), L. W., Lehmler(*), H. J., Kania-Korwel(*), I., Chahoud(*), I.; Schwarz(*), M.
Toxicol Sci, 133:29-41

Tags: Mass Spectrometry (Krause, E.)

Abstract: To assess the impact of a mixture containing dioxin-like and non-dioxin-like polychlorinated biphenyls (PCBs), male mice were initiated with N-nitroso-diethylamine and subsequently treated with PCB126, an Ah-Receptor agonist, and PCB153, acting via activation of the constitutive androstane receptor. The two congeners were given at two dose levels: the low dose was adjusted to induce similar to 150-fold increases in cytochrome P450 (Cyp)1a1 (PCB126) and Cyp2b10 mRNAs (PCB153), and the high dose was chosen as twice the low dose. To keep the liver PCB levels constant, mice were given initial loading doses followed by weekly maintenance doses calculated on the basis of the PCBs' half-lives. Mice were treated with the individual congeners (low and high dose) or with a mixture consisting of the low doses of the 2 PCBs. The following results were obtained: (1) the 2 PCBs produced dose-dependent increases in Cyp1a1 and Cyp2b10 mRNA, protein, and activity when given individually; (2) combined treatment caused more than additive effects on Cyp1a1 mRNA expression, protein level, and ethoxyresurofin activity; (3) changes in the levels of several proteins were detected by proteome analysis in livers of PCB-treated mice; (4) besides these biological responses, the individual PCBs caused no significant increase in the number of glucose-6-phospatase (G6Pase)-deficient neoplastic lesions in liver, whereas a moderate significant effect occurred in the combination group. These results suggest weak but significant response-additive effects of the 2 PCBs when given in combination. They also suggest that the Cyp biomarkers tend to overestimate the carcinogenic response produced by the PCBs in mouse liver.

Quantitative NMR analysis of Erk activity and inhibition by U0126 in a panel of patient-derived colorectal cancer cell lines
Rose, H. M., Stuiver, M., Thongwichian, R., Theillet, F. X., Feller(*), S. M.; Selenko, P.
Biochim Biophys Acta, 1834:1396-1401

Tags: In-Cell NMR (Selenko)

Abstract: We comparatively analyzed the basal activity of extra-cellular signal-regulated kinase (Erk1/2) in lysates of 10 human colorectal cancer cell lines by semi-quantitative Western blotting and time-resolved NMR spectroscopy. Both methods revealed heterogeneous levels of endogenous Erk1/2 activities in a highly consistent manner. Upon treatment with U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK) acting upstream of Erk1/2, Western-blotting and NMR congruently reported specific modulations of cellular phospho-Erk levels that translated into reduced kinase activities. Results obtained in this study highlight the complementary nature of antibody- and NMR-based phospho-detection techniques. They further exemplify the usefulness of time-resolved NMR measurements in providing fast and quantitative readouts of kinase activities and kinase inhibitor efficacies in native cellular environments. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Fast neurotransmitter release regulated by the endocytic scaffold intersectin
Sakaba(*), T., Kononenko, N. L., Bacetic, J., Pechstein, A., Schmoranzer, J., Yao(*), L., Barth(*), H., Shupliakov(*), O., Kobler(*), O., Aktories(*), K.; Haucke, V.
Proc Natl Acad Sci U S A, 110:8266-8271

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Sustained fast neurotransmission requires the rapid replenishment of release-ready synaptic vesicles (SVs) at presynaptic active zones. Although the machineries for exocytic fusion and for subsequent endocytic membrane retrieval have been well characterized, little is known about the mechanisms underlying the rapid recruitment of SVs to release sites. Here we show that the Down syndrome-associated endocytic scaffold protein intersectin 1 is a crucial factor for the recruitment of release-ready SVs. Genetic deletion of intersectin 1 expression or acute interference with intersectin function inhibited the replenishment of release-ready vesicles, resulting in short-term depression, without significantly affecting the rate of endocytic membrane retrieval. Acute perturbation experiments suggest that intersectin-mediated vesicle replenishment involves the association of intersectin with the fissioning enzyme dynamin and with the actin regulatory GTPase CDC42. Our data indicate a role for the endocytic scaffold intersectin in fast neurotransmitter release, which may be of prime importance for information processing in the brain.

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

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

What Goes around Comes around-A Comparative Study of the Influence of Chemical Modifications on the Antimicrobial Properties of Small Cyclic Peptides
Scheinpflug, K., Nikolenko, H., Komarov(*), I. V., Rautenbach(*), M.; Dathe, M.
Pharmaceuticals (Basel), 6:1130-1144

Tags: Peptide-Lipid-Interaction/ Peptide Transport (Dathe)

Abstract: Tryptophan and arginine-rich cyclic hexapeptides of the type cyclo-RRRWFW combine high antibacterial activity with rapid cell killing kinetics, but show low toxicity in human cell lines. The peptides fulfil the structural requirements for membrane interaction such as high amphipathicity and cationic charge, but membrane permeabilisation, which is the most common mode of action of antimicrobial peptides (AMPs), could not be observed. Our current studies focus on elucidating a putative membrane translocation mechanism whereupon the peptides might interfere with intracellular processes. These investigations require particular analytical tools: fluorescent analogues and peptides bearing appropriate reactive groups were synthesized and characterized in order to be used in confocal laser scanning microscopy and HPLC analysis. We found that minimal changes in both the cationic and hydrophobic domain of the peptides in most cases led to significant reduction of antimicrobial activity and/or changes in the mode of action. However, we were able to identify two modified peptides which exhibited properties similar to those of the cyclic parent hexapeptide and are suitable for subsequent studies on membrane translocation and uptake into bacterial cells.

Determination of glucan phosphorylation using heteronuclear 1H, 13C double and 1H, 13C, 31P triple-resonance NMR spectra
Schmieder, P., Nitschke(*), F., Steup(*), M., Mallow, K.; Specker, E.
Magn Reson Chem, 51:655-661

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

Abstract: Phosphorylation and dephosphorylation of starch and glycogen are important for their physicochemical properties and also their physiological functions. It is therefore desirable to reliably determine the phosphorylation sites. Heteronuclear multidimensional NMR-spectroscopy is in principle a straightforward analytical approach even for complex carbohydrate molecules. With heterogeneous samples from natural sources, however, the task becomes more difficult because a full assignment of the resonances of the carbohydrates is impossible to obtain. Here, we show that the combination of heteronuclear (1) H,(13) C and (1) H,(13) C,(31) P techniques and information derived from spectra of a set of reference compounds can lead to an unambiguous determination of the phosphorylation sites even in heterogeneous samples.

Functionalized 129Xe as a potential biosensor for membrane fluidity
Schnurr, M., Witte, C.; Schröder, L.
Phys Chem Chem Phys, 15:14178-14181

Tags: Molecular Imaging (Schröder)

Abstract: Using spin hyperpolarized xenon ((129)Xe) we investigate the impact of the local molecular environment on reversible host-guest interactions. We label Xe guest atoms that are temporarily bound to cryptophane-A hosts using the Hyper-CEST technique. By varying the length of the saturation pulse and utilizing an inverse Laplace transform we can determine depolarization times for the noble gas in different local environments, in this case biomembranes possessing different fluidity. We extend this technique to magnetic resonance imaging, mapping the spatial distribution of the different biomembranes. Such decays measured in biomembranes of 200 muM 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were characterized by mono-exponential decays with time constants of tauPOPC = 3.00(-0.61)(+0.77) s and tauDPPC(-4.16)(+5.19) = 22.15 s. Analyzing both environments simultaneously yielded a bi-exponential decay. This approach may give further insights into saturation transfer dynamics of reversibly bound Xe with applications extending into biomedical diagnostics.

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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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