FMP Publications

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

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[Professor Dr. sc. med.Walter Schilling (14th July 1932-7th April 2014)]
Grohe(*), C., Oehme(*), P.; Wiesner, B.
Pneumologie (Stuttgart, Germany), 68:432
(2014)

Tags: Cellular Imaging (Wiesner)

Differences between lutropin-mediated and choriogonadotropin-mediated receptor activation
Grzesik, P., Teichmann, A., Furkert, J., Rutz, C., Wiesner, B., Kleinau(*), G., Schülein, R., Gromoll(*), J.; Krause, G.
Febs J, 281:1479-1492
(2014)

Tags: Structural Bioinformatics and Protein Design (Krause, G.), Protein Trafficking (Schülein), Cellular Imaging (Wiesner)

Abstract: The human lutropin/choriogonadotropin receptor (hLHR) for the gonadotropic hormones human luteinizing hormone (hLH; lutropin) and human choriogonadotropin (hCG) is crucial for normal sexual development and fertility. We aimed to unravel differences between the two hLHR hormones in molecular activation mechanisms at hLHR. We utilized a specific hLHR variant that lacks exon 10 (hLHR-delExon10), which maintains full cAMP signaling by hCG, but decreases hLH-induced receptor signaling, resulting in a pathogenic phenotype. Exon 10 encodes 27 amino acids within the hinge region, which is an extracellular segment that is important for signaling and hormone interaction. Initially, we assumed that the lack of exon 10 might disturb intermolecular trans-activation of hLH, a mechanism that has been reported for hCG at hLHR. Coexpression of signaling-deficient hLHR and binding-deficient hLHR can be used to examine the mechanisms of receptor signaling, in particular intermolecular cooperation and intramolecular cis-activation. Therefore, hLHR-delExon10 was combined with the hLHR Lys605-->Glu mutant, in which signaling is abolished, and the hLHR mutant Cys131-->Arg, in which binding is deficient. We found that hCG signaling was partially rescued, indicating trans-activation. However, the hLH signal could not be restored via forced trans-activation with any construct. Fluorescence cross-correlation spectroscopy detected oligomerization in all combinations, indicating that these functional differences cannot be explained by monomerization of hLHR-delExon10. Thus, our data demonstrate not only that the different behavior of hLH at hLHR-delExon10 is unlikely to be related to modified intermolecular receptor activation, but also that hLH may exclusively stimulate the targeted hLHR by cis-activation, whereas hCG is also capable of inducing trans-activation.

Removal of albumin and immunoglobulins from canine cerebrospinal fluid using depletion kits: a feasibility study
Günther, R., Krause, E., Schümann, M., Ausseil(*), J., Heard(*), J. M., Blasig, I. E.; Haseloff, R. F.
Fluids and barriers of the CNS, 11:14
(2014)

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

Abstract: BACKGROUND: Highly abundant proteins in biological fluids such as serum or cerebrospinal fluid (CSF) can hinder the detection of proteins in lower abundance, e.g., potential biomarkers. Commercial products are available for the depletion of albumin and immunoglobulins (Igs), although most of these kits have not been validated for dog samples. The present study therefore examines the use of different types of depletion kits for dog CSF. FINDINGS: Three kits, with different mechanisms for the depletion of albumin and Igs, were tested with dog CSF specimens. One product significantly decreased the amount of albumin; with all kits, IgG was less efficiently removed than albumin. Mass spectrometry of the fractions eluted from the depletion columns revealed considerable co-depletion of other CSF proteins. CONCLUSIONS: A commercially available depletion kit was identified which depletes albumin and (to a lower extent) immunoglobulins from dog CSF. However, the limited efficacy and the concomitant loss of other proteins from the sample should be taken into account when using this product.

CD4+ T cells from human neonates and infants are poised spontaneously to run a nonclassical IL-4 program
Hebel(*), K., Weinert(*), S., Kuropka, B., Knolle(*), J., Kosak(*), B., Jorch(*), G., Arens(*), C., Krause, E., Braun-Dullaeus(*), R. C.; Brunner-Weinzierl(*), M. C.
J Immunol, 192:5160-5170
(2014)

Tags: Mass Spectrometry (Krause, E.)

Abstract: Senescence or biological aging impacts a vast variety of molecular and cellular processes. To date, it is unknown whether CD4(+) Th cells display an age-dependent bias for development into specific subpopulations. In this study, we show the appearance of a distinct CD4(+) T cell subset expressing IL-4 at an early stage of development in infant adenoids and cord blood that is lost during aging. We identified by flow cytometric, fluorescent microscopic, immunoblot, and mass spectrometric analysis a population of CD4(+) T cells that expressed an unglycosylated isoform of IL-4. This T cell subpopulation was found in neonatal but not in adult CD4(+) T cells. Furthermore, we show that the mRNA of the Th2 master transcription factor GATA3 is preferentially expressed in neonatal CD4(+) T cells. The Th2 phenotype of the IL-4(+)CD4(+) T cells could be reinforced in the presence of TGF-beta. Although the IL-4(+)CD4(+) T cells most likely originate from CD31(+)CD4(+) T recent thymic emigrants, CD31 was downregulated prior to secretion of IL-4. Notably, the secretion of IL-4 requires a so far unidentified trigger in neonatal T cells. This emphasizes that cytokine expression and secretion are differentially regulated processes. Our data support the hypothesis of an endogenously poised cytokine profile in neonates and suggest a link between cytokine production and the developmental stage of an organism. The determination of the IL-4 isoform-expressing cells in humans might allow the identification of Th2 precursor cells, which could provide novel intervention strategies directed against Th2-driven immunopathologies such as allergies.

Disrupting MLC1 and GlialCAM and ClC-2 interactions in leukodystrophy entails glial chloride channel dysfunction
Hoegg-Beiler, M. B., Sirisi(*), S., Orozco, I. J., Ferrer(*), I., Hohensee, S., Auberson, M., Gödde, K., Vilches(*), C., de Heredia(*), M. L., Nunes(*), V., Estevez(*), R.; Jentsch, T. J.
Nat Commun, 5:3475
(2014)

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Defects in the astrocytic membrane protein MLC1, the adhesion molecule GlialCAM or the chloride channel ClC-2 underlie human leukoencephalopathies. Whereas GlialCAM binds ClC-2 and MLC1, and modifies ClC-2 currents in vitro, no functional connections between MLC1 and ClC-2 are known. Here we investigate this by generating loss-of-function Glialcam and Mlc1 mouse models manifesting myelin vacuolization. We find that ClC-2 is unnecessary for MLC1 and GlialCAM localization in brain, whereas GlialCAM is important for targeting MLC1 and ClC-2 to specialized glial domains in vivo and for modifying ClC-2's biophysical properties specifically in oligodendrocytes (OLs), the cells chiefly affected by vacuolization. Unexpectedly, MLC1 is crucial for proper localization of GlialCAM and ClC-2, and for changing ClC-2 currents. Our data unmask an unforeseen functional relationship between MLC1 and ClC-2 in vivo, which is probably mediated by GlialCAM, and suggest that ClC-2 participates in the pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts.

Chemoselective Wittig and Michael Ligations of Unprotected Peptidyl Phosphoranes in Water Furnish Potent Inhibitors of Caspase-3
Holland-Nell, K., Fernandez-Bachiller, M. I., Ahsanullah, R.J.; Rademann, J.
Org Lett, 16:4428-4431
(2014)

Tags: Medicinal Chemistry (Rademann)

Abstract: Unprotected peptidyl phosphoranes 1 with sequence Ac-L-aspartyl-L-glutamyl-L-valinyl-L-aspartyl are released from polymer support and react with aliphatic and aromatic aldehydes in aqueous medium in a Wittig ligation. Obtained vinyl ketones 6-12 are potent inhibitors of caspase-3. Vinyl ketone 6, derived from formaldehyde, undergoes Michael ligations with thiol nucleophiles furnishing products 14-16, also in aqueous medium. The demonstrated ligation reactions enable the modification of complex functionalized peptides in water providing bioactive protein ligands without side-chain protection.

Design of a General-Purpose European Compound Screening Library for EU-OPENSCREEN
Horvath(*), D., Lisurek, M., Rupp, B., Kühne, R., Specker, E., von Kries, J., Rognan(*), D., Andersson(*), C. D., Almqvist(*), F., Elofsson(*), M., Enqvist(*), P. A., Gustavsson(*), A. L., Remez(*), N., Mestres(*), J., Marcou(*), G., Varnek(*), A., Hibert(*), M., Quintana(*), J.; Frank, R.
Chemmedchem, 9:2309-2326
(2014)

Tags: Chemical Systems Biology (Frank), Screening Unit (von Kries), Computational Chemistry and Protein Design (Kühne)

Abstract: This work describes a collaborative effort to define and apply a protocol for the rational selection of a general-purpose screening library, to be used by the screening platforms affiliated with the EU-OPENSCREEN initiative. It is designed as a standard source of compounds for primary screening against novel biological targets, at the request of research partners. Given the general nature of the potential applications of this compound collection, the focus of the selection strategy lies on ensuring chemical stability, absence of reactive compounds, screening-compliant physicochemical properties, loose compliance to drug-likeness criteria (as drug design is a major, but not exclusive application), and maximal diversity/coverage of chemical space, aimed at providing hits for a wide spectrum of drugable targets. Finally, practical availability/cost issues cannot be avoided. The main goal of this publication is to inform potential future users of this library about its conception, sources, and characteristics. The outline of the selection procedure, notably of the filtering rules designed by a large committee of European medicinal chemists and chemoinformaticians, may be of general methodological interest for the screening/medicinal chemistry community. The selection task of 200K molecules out of a pre-filtered set of 1.4M candidates was shared by five independent European research groups, each picking a subset of 40K compounds according to their own in-house methodology and expertise. An in-depth analysis of chemical space coverage of the library serves not only to characterize the collection, but also to compare the various chemoinformatics-driven selection procedures of maximal diversity sets. Compound selections contributed by various participating groups were mapped onto general-purpose self-organizing maps (SOMs) built on the basis of marketed drugs and bioactive reference molecules. In this way, the occupancy of chemical space by the EU-OPENSCREEN library could be directly compared with distributions of known bioactives of various classes. This mapping highlights the relevance of the selection and shows how the consensus reached by merging the five different 40K selections contributes to achieve this relevance. The approach also allows one to readily identify subsets of target-or target-class-oriented compounds from the EU-OPENSCREEN library to suit the needs of the diverse range of potential users. The final EU-OPENSCREEN library, assembled by merging five independent selections of 40K compounds from various expert groups, represents an excellent example of a Europe-wide collaborative effort toward the common objective of building best-in-class European open screening platforms.

Real-Time Monitoring of Membrane-Protein Reconstitution by Isothermal Titration Calorimetry
Jahnke, N., Krylova, O. O., Hoomann(*), T., Vargas(*), C., Fiedler(*), S., Pohl(*), P.; Keller(*), S.
Anal Chem, 86:920-927
(2014)

Tags: Biophysics of Membrane Proteins (Keller)

Abstract: Phase diagrams offer a wealth of thermodynamic information on aqueous mixtures of bilayer-forming lipids and micelle-forming detergents, providing a straightforward means of monitoring and adjusting the supramolecular state of such systems. However, equilibrium phase diagrams are of very limited use for the reconstitution of membrane proteins because of the occurrence of irreversible, unproductive processes such as aggregation and precipitation that compete with productive reconstitution. Here, we exemplify this by dissecting the effects of the K+ channel KcsA on the process of bilayer self-assembly in a mixture of Escherichia coli polar lipid extract and the nonionic detergent octyl-beta-D-glucopyranoside. Even at starting concentrations in the low micromolar range, KcsA has a tremendous impact on the supramolecular organization of the system, shifting the critical lipid/detergent ratios at the onset and completion of vesicle formation by more than 2-fold. Thus, equilibrium phase diagrams obtained for protein-free lipid/detergent mixtures would be misleading when used to guide the reconstitution process. To address this issue, we demonstrate that, even under such nonequilibrium conditions, high-sensitivity isothermal titration calorimetry can be exploited to monitor the progress of membrane-protein reconstitution in real time, in a noninvasive manner, and at high resolution to yield functional proteoliposomes with a narrow size distribution for further downstream applications.

Low-power polarization transfer between deuterons and spin-1/2 nuclei using adiabatic (CP)-C-RESPIRATION in solid-state NMR
Jain(*), S. K., Nielsen(*), A. B., Hiller, M., Handel, L., Ernst(*), M., Oschkinat, H., Akbey, Ü.; Nielsen(*), N. C.
Physical Chemistry Chemical Physics, 16:2827-2830
(2014)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Establishing high-resolution structures of biological macromolecules in heterogeneous environments by MAS solid-state NMR is an important challenge where development of advanced experimental procedures is in high demand. Promising new methods take advantage of samples with extensive H-2, C-13, and N-15 isotope labelling, effectively diluting 1H spins. In many cases, a sufficient amount of H-1 at exchangeable sites cannot be re-established during the purification procedure, hence it is necessary to exploit also the potential of H-2 as a starting point in pulse sequences, capitalizing on its short T-1 as compared to C-13, and to detect carbon or proton spins as appropriate. Here we present a new method that enables the required high-efficiency H-2, C-13, and N-15 polarization transfer to be accomplished under the limited H-2 rf power conditions using current H-1, H-2, C-13 and N-15 quadruple-resonance MAS NMR instrumentation.

GlialCAM, a CLC-2 Cl(-) channel subunit, activates the slow gate of CLC chloride channels
Jeworutzki(*), E., Lagostena(*), L., Elorza-Vidal(*), X., Lopez-Hernandez, T., Estevez(*), R.; Pusch(*), M.
Biophys J, 107:1105-1116
(2014)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: GlialCAM, a glial cell adhesion molecule mutated in megalencephalic leukoencephalopathy with subcortical cysts, targets the CLC-2 Cl(-) channel to cell contacts in glia and activates CLC-2 currents in vitro and in vivo. We found that GlialCAM clusters all CLC channels at cell contacts in vitro and thus studied GlialCAM interaction with CLC channels to investigate the mechanism of functional activation. GlialCAM slowed deactivation kinetics of CLC-Ka/barttin channels and increased CLC-0 currents opening the common gate and slowing its deactivation. No functional effect was seen for common gate deficient CLC-0 mutants. Similarly, GlialCAM targets the common gate deficient CLC-2 mutant E211V/H816A to cell contacts, without altering its function. Thus, GlialCAM is able to interact with all CLC channels tested, targeting them to cell junctions and activating them by stabilizing the open configuration of the common gate. These results are important to better understand the physiological role of GlialCAM/CLC-2 interaction.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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