FMP Publications

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

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References
Narrow carbonyl resonances in proton-diluted proteins facilitate NMR assignments in the solid-state
Linser, R., Fink, U.; Reif, B.
J. Biomol. NMR, 47:1-6
(2010)

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: HNCO/HNCACO type correlation experiments are an alternative for assignment of backbone resonances in extensively deuterated proteins in the solid-state, given the fact that line widths on the order of 14-17 Hz are achieved in the carbonyl dimension without the need of high power decoupling. The achieved resolution demonstrates that MAS solid-state NMR on extensively deuterated proteins is able to compete with solution-state NMR spectroscopy if proteins are investigated with correlation times tau (c) that exceed 25 ns.

Simultaneous detection of protein phosphorylation and acetylation by high-resolution NMR spectroscopy
Liokatis, S., Dose, A., Schwarzer, D.; Selenko, P.
J Am Chem Soc, 132:14704-14705
(2010)

Tags: In-Cell NMR (Selenko), Protein Chemistry (Schwarzer)

Abstract: Post-translational protein modifications (PTMs) such as phosphorylation and acetylation regulate a large number of eukaryotic signaling processes. In most instances, it is the combination of different PTMs that "encode" the biological outcome of these covalent amendments in a highly dynamic and cell-state-specific manner. Most research tools fail to detect different PTMs in a single experiment and are unable to directly observe dynamic PTM states in complex environments such as cell extracts or intact cells. Here we describe in situ observations of phosphorylation and acetylation reactions by high-resolution liquid-state NMR spectroscopy. We delineate the NMR characteristics of progressive lysine acetylation and provide in vitro examples of joint phosphorylation and acetylation events and how they can be deciphered on a residue-specific basis and in a time-resolved and quantitative manner. Finally, we extend our NMR investigations to cellular phosphorylation and acetylation events in human cell extracts and demonstrate the unique ability of NMR spectroscopy to simultaneously report the establishment of these PTMs by endogenous cellular enzymes.

Design of chemical libraries with potentially bioactive molecules applying a maximum common substructure concept
Lisurek, M., Rupp, B., Wichard, J., Neuenschwander, M., von Kries, J. P., Frank(*), R., Rademann, J.; Kühne, R.
Mol Divers, 14:401-408
(2010)

Tags: Screening Unit (von Kries), Medicinal Chemistry (Rademann), Computational Chemistry/Drug Design (Kühne)

Abstract: Success in small molecule screening relies heavily on the preselection of compounds. Here, we present a strategy for the enrichment of chemical libraries with potentially bioactive compounds integrating the collected knowledge of medicinal chemistry. Employing a genetic algorithm, substructures typically occurring in bioactive compounds were identified using the World Drug Index. Availability of compounds containing the selected substructures was analysed in vendor libraries, and the substructure-specific sublibraries were assembled. Compounds containing reactive, undesired functional groups were omitted. Using a diversity filter for both physico-chemical properties and the substructure composition, the compounds of all the sublibraries were ranked. Accordingly, a screening collection of 16,671 compounds was selected. Diversity and chemical space coverage of the collection indicate that it is highly diverse and well-placed in the chemical space spanned by bioactive compounds. Furthermore, secondary assay-validated hits presented in this study show the practical relevance of our library design strategy.

A novel subtype of AP-1-binding motif within the palmitoylated trans-Golgi network/endosomal accessory protein Gadkin/gamma-BAR
Maritzen(*), T., Schmidt(*), M. R., Kukhtina(*), V., Higman, V. A., Strauss, H., Volkmer(*), R., Oschkinat, H., Dotti(*), C. G.; Haucke, V.
J Biol Chem, 285:4074-4086
(2010)

Tags: Molecular Pharmacology and Cell Biology (Haucke), Protein Structure (Oschkinat)

Abstract: Membrane traffic between the trans-Golgi network (TGN) and endosomes is mediated in part by the assembly of clathrin-AP-1 adaptor complex-coated vesicles. This process involves multiple accessory proteins that directly bind to the ear domain of AP-1gamma via degenerate peptide motifs that conform to the consensus sequence diameterG(P/D/E)(diameter/L/M) (with diameter being a large hydrophobic amino acid). Recently, gamma-BAR (hereafter referred to as Gadkin for reasons explained below) has been identified as a novel AP-1 recruitment factor involved in AP-1-dependent endosomal trafficking of lysosomal enzymes. How precisely Gadkin interacts with membranes and with AP-1gamma has remained unclear. Here we show that Gadkin is an S-palmitoylated peripheral membrane protein that lacks stable tertiary structure. S-Palmitoylation is required for the recruitment of Gadkin to TGN/endosomal membranes but not for binding to AP-1. Furthermore, we identify a novel subtype of AP-1-binding motif within Gadkin that specifically associates with the gamma1-adaptin ear domain. Mutational inactivation of this novel type of motif, either alone or in combination with three more conventional AP-1gamma binding peptides, causes Gadkin to mislocalize to the plasma membrane and interferes with its ability to render AP-1 brefeldin A-resistant, indicating its physiological importance. Our studies thus unravel the molecular basis for Gadkin-mediated AP-1 recruitment to TGN/endosomal membranes and identify a novel subtype of the AP-1-binding motif.

Segmental expression of claudin proteins correlates with tight junction barrier properties in rat intestine
Markov(*), A. G., Veshnyakova, A., Fromm(*), M., Amasheh(*), M.; Amasheh(*), S.
J Comp Physiol B, 180:591-598
(2010)

Tags: Structural Bioinformatics and Protein Design (Krause, G.)

Abstract: In tubular epithelia, barrier function varies in a segment-specific way. The aim of this study was to correlate the presence of tight junction proteins and paracellular barrier properties along rat intestine. Tissue segments of duodenum, jejunum, ileum, and colon were stripped of submucosal cell layers and mounted in Ussing chambers for impedance spectroscopy to measure epithelial resistance (R (epi)). In parallel, expression of tight junction proteins was analysed by Western blots and immune fluorescence confocal microscopy. Colon showed highest R (epi), followed by duodenum, jejunum, and ileum. In small intestine, common transepithelial resistance (R (trans) or TER) overestimated true R (epi) by similar to 60%. In colon, strongest expression of "tightening" claudins 1, 3, 4, 5, and 8 was detected. In accordance with R (epi) the most proximal of the small intestinal segments, duodenum exhibited highest expression of "tightening" claudins and lowest expression of claudins mediating permeability, namely claudin-2, -7, and -12, compared to jejunum and ileum. These results correspond to the specific role of the duodenum as the first segment facing the acidic gastric content.

Routes of epithelial water flow: aquaporins versus cotransporters
Mollajew, R., Zocher(*), F., Horner(*), A., Wiesner, B., Klussmann, E.; Pohl, P.
Biophys J, 99:3647-3656
(2010)

Tags: Anchored Signalling (Klussmann), Cellular Imaging (Wiesner)

Abstract: The routes water takes through membrane barriers is still a matter of debate. Although aquaporins only allow transmembrane water movement along an osmotic gradient, cotransporters are believed to be capable of water transport against the osmotic gradient. Here we show that the renal potassium-chloride-cotransporter (KCC1) does not pump a fixed amount of water molecules per movement of one K(+) and one Cl(-), as was reported for the analogous transporter in the choroid plexus. We monitored water and potassium fluxes through monolayers of primary cultured renal epithelial cells by detecting tiny solute concentration changes in the immediate vicinity of the monolayer. KCC1 extruded K(+) ions in the presence of a transepithelial K(+) gradient, but did not transport water. KCC1 inhibition reduced epithelial osmotic water permeability P(f) by roughly one-third, i.e., the effect of inhibitors was small in resting cells and substantial in hormonal stimulated cells that contained high concentrations of aquaporin-2 in their apical membranes. The furosemide or DIOA (dihydroindenyl-oxy-alkanoic acid)-sensitive water flux was much larger than expected when water passively followed the KCC1-mediated ion flow. The inhibitory effect of these drugs on water flux was reversed by the K(+)-H(+) exchanger nigericin, indicating that KCC1 affects water transport solely by K(+) extrusion. Intracellular K(+) retention conceivably leads to cell swelling, followed by an increased rate of endocytic AQP2 retrieval from the apical membrane.

The late endosomal ClC-6 mediates proton/chloride countertransport in heterologous plasma membrane expression
Neagoe, I., Stauber, T., Fidzinski, P., Bergsdorf, E. Y.; Jentsch, T. J.
J Biol Chem, 285:21689-21697
(2010)

Tags: Physiology and Pathology of Ion Transport (Jentsch

Abstract: Members of the CLC protein family of Cl(-) channels and transporters display the remarkable ability to function as either chloride channels or Cl(-)/H(+) antiporters. Due to the intracellular localization of ClC-6 and ClC-7, it has not yet been possible to study the biophysical properties of these members of the late endosomal/lysosomal CLC branch in heterologous expression. Whereas recent data suggest that ClC-7 functions as an antiporter, transport characteristics of ClC-6 have remained entirely unknown. Here, we report that fusing the green fluorescent protein (GFP) to the N terminus of ClC-6 increased its cell surface expression, allowing us to functionally characterize ClC-6. Compatible with ClC-6 mediating Cl(-)/H(+) exchange, Xenopus oocytes expressing GFP-tagged ClC-6 alkalinized upon depolarization. This alkalinization was dependent on the presence of extracellular anions and could occur against an electrochemical proton gradient. As observed in other CLC exchangers, ClC-6-mediated H(+) transport was abolished by mutations in either the "gating" or "proton" glutamate. Overexpression of GFP-tagged ClC-6 in CHO cells elicited small, outwardly rectifying currents with a Cl(-) > I(-) conductance sequence. Mutating the gating glutamate of ClC-6 yielded an ohmic anion conductance that was increased by additionally mutating the "anion-coordinating" tyrosine. Additionally changing the chloride-coordinating serine 157 to proline increased the NO(3)(-) conductance of this mutant. Taken together, these data demonstrate for the first time that ClC-6 is a Cl(-)/H(+) antiporter.

Reciprocal regulation of aquaporin-2 abundance and degradation by protein kinase A and p38-MAP kinase
Nedvetsky, P. I., Tabor, V., Tamma(*), G., Beulshausen, S., Skroblin, P., Kirschner, A., Mutig(*), K., Boltzen, M., Petrucci, O., Vossenkamper, A., Wiesner, B., Bachmann(*), S., Rosenthal(*), W.; Klussmann, E.
Journal of the American Society of Nephrology : JASN, 21:1645-1656
(2010)

Tags: Anchored Signalling (Klussmann), Cellular Imaging (Wiesner)

Abstract: Arginine-vasopressin (AVP) modulates the water channel aquaporin-2 (AQP2) in the renal collecting duct to maintain homeostasis of body water. AVP binds to vasopressin V2 receptors (V2R), increasing cAMP, which promotes the redistribution of AQP2 from intracellular vesicles into the plasma membrane. cAMP also increases AQP2 transcription, but whether altered degradation also modulates AQP2 protein levels is not well understood. Here, elevation of cAMP increased AQP2 protein levels within 30 minutes in primary inner medullary collecting duct (IMCD) cells, in human embryonic kidney (HEK) 293 cells ectopically expressing AQP2, and in mouse kidneys. Accelerated transcription or translation did not explain this increase in AQP2 abundance. In IMCD cells, cAMP inhibited p38-mitogen-activated protein kinase (p38-MAPK) via activation of protein kinase A (PKA). Inhibition of p38-MAPK associated with decreased phosphorylation (serine 261) and polyubiquitination of AQP2, preventing proteasomal degradation. Our results demonstrate that AVP enhances AQP2 protein abundance by altering its proteasomal degradation through a PKA- and p38-MAPK-dependent pathway.

Azides Derived from Colchicine and their Use in Library Synthesis: a Practical Entry to New Bioactive Derivatives of an Old Natural Drug
Nicolaus(*), N., Zapke, J., Riesterer(*), P., Neudörfl, J. M., Prokop(*), A., Oschkinat, H.; Schmalz(*), H. G.
Chemmedchem, 5:661-665
(2010)

Tags: Protein Structure (Oschkinat)

No evidence for a role of CLCN2 variants in idiopathic generalized epilepsy
Niemeyer(*), M. I., Cid(*), L. P., Sepulveda(*), F. V., Blanz(*), J., Auberson(*), M.; Jentsch, T. J.
Nat Genet, 42:3-3
(2010)

Tags: Physiology and Pathology of Ion Transport (Jentsch

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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
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info(at)fmp-berlin.de

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