FMP Publications

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

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Antifungal membranolytic activity of the tyrocidines against filamentous plant fungi
Rautenbach(*), M., Troskie(*), A. M., Vosloo(*), J. A.; Dathe, M. E.
Biochimie, 130:122-131

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

Abstract: The tyrocidines and analogues are cyclic decapeptides produced by Brevibacillus parabrevis with a conserved sequence of cyclo(D-Phe1-Pro2-X3-x4-Asn5-Gln6-X7-Val8-X9-Leu10) with Trp3,4/Phe3,4 in the aromatic dipeptide unit, Lys9/Orn9 as their cationic residue and Tyr (tyrocidines), Trp (tryptocidines) or Phe (phenicidines) in position 7. Previous studies indicated they have a broad antifungal spectrum with the peptides containing a Tyr residue in position 7 being more active than those with a Phe or Trp residue in this position. Detailed analysis of antifungal inhibition parameters revealed that Phe3-D-Phe4 in the aromatic dipeptide unit lead to more consistent activity against the three filamentous fungi in this study. These peptides exhibited high membrane activity and fast leakage kinetics against model membranes emulating fungal membranes, with selectivity towards ergosterol containing membranes. More fluid membranes and doping of liposomes with the sphingolipid, glucosylceramide, led to a decreased permeabilising activity. Peptide-induced uptake of membrane impermeable dyes was observed in hyphae of both Fusarium solani and Botrytis cinerea, with uptake more pronounced at the hyphal growth tips that are known to contain ergosterol-sphigolipid rich lipid rafts. Tyrocidine interaction with these rafts may lead to the previously observed fungal hyperbranching. However, the leakage of model membranes and Bot. cinerea did not correlate directly with the antifungal inhibition parameters, indicating another target or mode of action. Proteinase K treatment of target fungi had a minimal influence or even improved the tyrocidine activity, ruling out a mannoprotein target in the fungal cell wall. beta-glucanase treatment of Bot. cinerea did not significantly affect the tyrocidine activity, but there was a significant loss in activity towards the beta-glucanase treated F. solani. This study showed the tyrocidine antifungal membrane activity is selective towards ergosterol and possibly lipid rafts, but also point to additional targets such as the cell wall beta-glucans that could modulate their activity.

VRAC: molecular identification as LRRC8 heteromers with differential functions
Jentsch, T. J., Lutter, D., Planells-Cases, R., Ullrich, F.; Voss, F. K.
Pflugers Arch, 468:385-393

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: A major player of vertebrate cell volume regulation is the volume-regulated anion channel (VRAC), which conducts halide ions and organic osmolytes to counteract osmotic imbalances. The molecular entity of this channel was unknown until very recently, although its biophysical characteristics and diverse physiological roles have been extensively studied over the last 30 years. On the road to the molecular identification of VRAC, experimental difficulties led to the proposal of a variety of false candidates. In 2014, in a final breakthrough, two groups independently identified LRRC8A as indispensable component of VRAC. LRRC8A is part of the leucine-rich repeat containing 8 family, which is comprised of five members (LRRC8A-E). Of those, LRRC8A is an obligatory subunit of VRAC but it needs at least one of the other family members to mediate the swelling-induced Cl(-) current ICl,vol. This review discusses the remarkable journey which led to the molecular identification of VRAC, evidence for LRRC8 proteins forming the VRAC pore and their heteromeric assembly. Furthermore, first major insights on the role of LRRC8 proteins in cancer drug resistance and apoptosis and the role of LRRC8D in cisplatin and taurine transport will be summarized.

Inactivation and Anion Selectivity of Volume-regulated Anion Channels (VRACs) Depend on C-terminal Residues of the First Extracellular Loop
Ullrich, F., Reincke, S. M., Voss, F. K., Stauber, T.; Jentsch, T. J.
J Biol Chem, 291:17040-17048

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Canonical volume-regulated anion channels (VRACs) are crucial for cell volume regulation and have many other important roles, including tumor drug resistance and release of neurotransmitters. Although VRAC-mediated swelling-activated chloride currents (ICl,vol) have been studied for decades, exploration of the structure-function relationship of VRAC has become possible only after the recent discovery that VRACs are formed by differently composed heteromers of LRRC8 proteins. Inactivation of ICl,vol at positive potentials, a typical hallmark of VRACs, strongly varies between native cell types. Exploiting the large differences in inactivation between different LRRC8 heteromers, we now used chimeras assembled from isoforms LRRC8C and LRRC8E to uncover a highly conserved extracellular region preceding the second LRRC8 transmembrane domain as a major determinant of ICl,vol inactivation. Point mutations identified two amino acids (Lys-98 and Asp-100 in LRRC8A and equivalent residues in LRRC8C and -E), which upon charge reversal strongly altered the kinetics and voltage dependence of inactivation. Importantly, charge reversal at the first position also reduced the iodide > chloride permeability of ICl,vol This change in selectivity was stronger when both the obligatory LRRC8A subunit and the other co-expressed isoform (LRR8C or -E) carried such mutations. Hence, the C-terminal part of the first extracellular loop not only determines VRAC inactivation but might also participate in forming its outer pore. Inactivation of VRACs may involve a closure of the extracellular mouth of the permeation pathway.

Tubular Epithelial NF-kappaB Activity Regulates Ischemic AKI
Marko(*), L., Vigolo(*), E., Hinze(*), C., Park(*), J. K., Roel(*), G., Balogh(*), A., Choi(*), M., Wübken(*), A., Cording, J., Blasig, I. E., Luft(*), F. C., Scheidereit(*), C., Schmidt-Ott(*), K. M., Schmidt-Ullrich(*), R.; Müller(*), D. N.
Journal of the American Society of Nephrology : JASN, 27:2658-2669

Tags: Molecular Cell Physiology (Blasig, I.E.)

Abstract: NF-kappaB is a key regulator of innate and adaptive immunity and is implicated in the pathogenesis of AKI. The cell type-specific functions of NF-kappaB in the kidney are unknown; however, the pathway serves distinct functions in immune and tissue parenchymal cells. We analyzed tubular epithelial-specific NF-kappaB signaling in a mouse model of ischemia-reperfusion injury (IRI)-induced AKI. NF-kappaB reporter activity and nuclear localization of phosphorylated NF-kappaB subunit p65 analyses in mice revealed that IRI induced widespread NF-kappaB activation in renal tubular epithelia and in interstitial cells that peaked 2-3 days after injury. To genetically antagonize tubular epithelial NF-kappaB activity, we generated mice expressing the human NF-kappaB super-repressor IkappaBalphaDeltaN in renal proximal, distal, and collecting duct epithelial cells. Compared with control mice, these mice exhibited improved renal function, reduced tubular apoptosis, and attenuated neutrophil and macrophage infiltration after IRI-induced AKI. Furthermore, tubular NF-kappaB-dependent gene expression profiles revealed temporally distinct functional gene clusters for apoptosis, chemotaxis, and morphogenesis. Primary proximal tubular cells isolated from IkappaBalphaDeltaN-expressing mice and exposed to hypoxia-mimetic agent cobalt chloride exhibited less apoptosis and expressed lower levels of chemokines than cells from control mice did. Our results indicate that postischemic NF-kappaB activation in renal tubular epithelia aggravates tubular injury and exacerbates a maladaptive inflammatory response.

RIM-binding protein 2 regulates release probability by fine-tuning calcium channel localization at murine hippocampal synapses
Grauel(*), M. K., Maglione, M., Reddy-Alla(*), S., Willmes(*), C. G., Brockmann(*), M. M., Trimbuch(*), T., Rosenmund(*), T., Pangalos(*), M., Vardar(*), G., Stumpf(*), A., Walter, A. M., Rost(*), B. R., Eickholt(*), B. J., Haucke, V., Schmitz(*), D., Sigrist(*), S. J.; Rosenmund(*), C.
Proc Natl Acad Sci U S A, 113:11615-11620

Tags: Molecular Pharmacology and Cell Biology (Haucke), Molecular and Theoretical Neuroscience (Walter)

Abstract: The tight spatial coupling of synaptic vesicles and voltage-gated Ca2+ channels (CaVs) ensures efficient action potential-triggered neurotransmitter release from presynaptic active zones (AZs). Rab-interacting molecule-binding proteins (RIM-BPs) interact with Ca2+ channels and via RIM with other components of the release machinery. Although human RIM-BPs have been implicated in autism spectrum disorders, little is known about the role of mammalian RIM-BPs in synaptic transmission. We investigated RIM-BP2-deficient murine hippocampal neurons in cultures and slices. Short-term facilitation is significantly enhanced in both model systems. Detailed analysis in culture revealed a reduction in initial release probability, which presumably underlies the increased short-term facilitation. Superresolution microscopy revealed an impairment in CaV2.1 clustering at AZs, which likely alters Ca2+ nanodomains at release sites and thereby affects release probability. Additional deletion of RIM-BP1 does not exacerbate the phenotype, indicating that RIM-BP2 is the dominating RIM-BP isoform at these synapses.

Progesterone, estrogen, and androgen receptors in the corpus luteum of the domestic cat, Iberian lynx (Lynx pardinus) and Eurasian lynx (Lynx lynx)
Amelkina(*), O., Zschockelt(*), L., Painer(*), J., Serra(*), R., Villaespesa(*), F., Krause, E., Jewgenow(*), K.; Braun(*), B. C.
Theriogenology, 86:2107-2118

Tags: Mass Spectrometry (Krause, E.)

Abstract: In contrast to the species studied, the corpus luteum (CL) of Iberian and Eurasian lynx physiologically persists in the ovary for more than 2 years and continues to secrete progesterone. Such persistent CL (perCL) transition into the next cycle and are present in the ovary together with the freshly formed CL (frCL) of a new ovulation. To date, the mechanisms supporting such CL persistence are not known. We analyzed the potential receptivity of feline CL to sex steroids through mRNA measurements of progesterone receptor (PGR), progesterone receptor membrane components (PGRMC) 1 and 2, estrogen receptor (ESR) 1 and ESR2, G protein-coupled estrogen receptor 1 (GPER1), and androgen receptor (AR). All receptors were present in domestic cat CL during pregnancy and the nonpregnant luteal phase, in frCL and perCL of post-mating Iberian lynx and in perCL of pre-mating Eurasian lynx. Mass spectrometry detected the presence of PGRMC1 protein in frCL and perCL of the Iberian lynx. In both domestic cat and lynx CL, PGR, PGRMC1, and ESR1 proteins were localized in luteal cells by immunohistochemistry. The mRNA levels of PGR, PGRMC1, PGRMC2, ESR1, and AR changed significantly throughout the domestic cat luteal phase. This may indicate involvement of these receptors in the processes of formation, maintenance, and regression of feline CL. In Iberian lynx, expression of PGRMC1, PGRCM2, ESR1, GPER1, and AR was significantly higher in perCL compared with frCL, whereas ESR2 was reversed. High mRNA amounts of these receptors in perCL suggest that physiological persistence of lynx CL may be partly regulated by actions of sex steroids through their nuclear and/or membrane receptors.

Urolinin: The First Linear Peptidic Urotensin-II Receptor Agonist
Bandholtz(*), S., Erdmann(*), S., von Hacht(*), J. L., Exner(*), S., Krause, G., Kleinau(*), G.; Grotzinger(*), C.
Journal of medicinal chemistry, 59:10100-10112

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

Abstract: This study investigated the role of individual U-II amino acid positions and side chain characteristics important for U-IIR activation. A complete permutation library of 209 U-II variants was studied in an activity screen that contained single substitution variants of each position with one of the other 19 proteinogenic amino acids. Receptor activation was measured using a cell-based high-throughput fluorescence calcium mobilization assay. We generated the first complete U-II substitution map for U-II receptor activation, resulting in a detailed view into the structural features required for receptor activation, accompanied by complementary information from receptor modeling and ligand docking studies. On the basis of the systematic SAR study of U-II, we created 33 further short and linear U-II variants from eight to three amino acids in length, including d- and other non-natural amino acids. We identified the first high-potency linear U-II analogues. Urolinin, a linear U-II agonist (nWWK-Tyr(3-NO2)-Abu), shows low nanomolar potency as well as improved metabolic stability.

Reversible Opening of Intercellular Junctions of Intestinal Epithelial and Brain Endothelial Cells With Tight Junction Modulator Peptides
Bocsik(*), A., Walter(*), F. R., Gyebrovszki(*), A., Fulop(*), L., Blasig, I., Dabrowski, S., Otvos(*), F., Toth(*), A., Rakhely(*), G., Veszelka(*), S., Vastag(*), M., Szabo-Revesz(*), P.; Deli(*), M. A.
Journal of pharmaceutical sciences, 105:754-765

Tags: Molecular Cell Physiology (Blasig, I.E.)

Abstract: The intercellular junctions restrict the free passage of hydrophilic compounds through the paracellular clefts. Reversible opening of the tight junctions of biological barriers is investigated as one of the ways to increase drug delivery to the systemic circulation or the central nervous system. Six peptides, ADT-6, HAV-6, C-CPE, 7-mer (FDFWITP, PN-78), AT-1002, and PN-159, acting on different integral membrane and linker junctional proteins were tested on Caco-2 intestinal epithelial cell line and a coculture model of the blood-brain barrier. All peptides tested in nontoxic concentrations showed a reversible tight junctions modulating effect and were effective to open the paracellular pathway for the marker molecules fluorescein and albumin. The change in the structure of cell-cell junctions was verified by immunostaining for occludin, claudin-4,-5, ZO-1, beta-catenin, and E-cadherin. Expression levels of occludin and claudins were measured in both models. We could demonstrate a selectivity of C-CPE, ADT-6, and HAV-6 peptides for epithelial cells and 7-mer and AT-1002 peptides for brain endothelial cells. PN-159 was the most effective modulator of junctional permeability in both models possibly acting via claudin-1 and -5. Our results indicate that these peptides can be effectively and selectively used as potential pharmaceutical excipients to improve drug delivery across biological barriers.

The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6
Boschert(*), V., Frisch(*), C., Back(*), J. W., van Pee(*), K., Weidauer(*), S. E., Muth(*), E. M., Schmieder, P., Beerbaum, M., Knappik(*), A., Timmerman(*), P.; Mueller(*), T. D.
Open biology, 6

Tags: Solution NMR (Schmieder)

Abstract: The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure-function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis.

Cellular Cations Control Conformational Switching of Inositol Pyrophosphate Analogues
Hager(*), A., Wu(*), M., Wang(*), H., Brown, N. W., Jr., Shears(*), S. B., Veiga(*), N.; Fiedler, D.
Chemistry, 22:12406-12414

Tags: Chemical Biology I (Fiedler)

Abstract: The inositol pyrophosphate messengers (PP-InsPs) are emerging as an important class of cellular regulators. These molecules have been linked to numerous biological processes, including insulin secretion and cancer cell migration, but how they trigger such a wide range of cellular responses has remained unanswered in many cases. Here, we show that the PP-InsPs exhibit complex speciation behaviour and propose that a unique conformational switching mechanism could contribute to their multifunctional effects. We synthesised non-hydrolysable bisphosphonate analogues and crystallised the analogues in complex with mammalian PPIP5K2 kinase. Subsequently, the bisphosphonate analogues were used to investigate the protonation sequence, metal-coordination properties, and conformation in solution. Remarkably, the presence of potassium and magnesium ions enabled the analogues to adopt two different conformations near physiological pH. Understanding how the intrinsic chemical properties of the PP-InsPs can contribute to their complex signalling outputs will be essential to elucidate their regulatory functions.

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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 
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