FMP Publications

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

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Genetic deficiency in neprilysin or its pharmacological inhibition initiate excessive stress-induced alcohol consumption in mice
Maul, B., Becker, M., Gembardt(*), F., Becker(*), A., Schultheiss(*), H. P., Siems, W. E.; Walther(*), T.
Plos One, 7:e50187

Tags: Biochemical Neurobiology (Siems)

Abstract: Both acquired and inherited genetic factors contribute to excessive alcohol consumption and the corresponding development of addiction. Here we show that the genetic deficiency in neprilysin [NEP] did not change the kinetics of alcohol degradation but led to an increase in alcohol intake in mice in a 2-bottle-free-choice paradigm after one single stress stimulus (intruder). A repetition of such stress led to an irreversible elevated alcohol consumption. This phenomenon could be also observed in wild-type mice receiving an orally active NEP inhibitor. We therefore elucidated the stress behavior in NEP-deficient mice. In an Elevated Plus Maze, NEP knockouts crossed more often the area between the arms, implicating a significant stronger stress response. Furthermore, such animals showed a decreased locomotor activity under intense light in a locomotor activity test, identifying such mice to be more responsive in aversive situations than their wild-type controls. Since the reduction in NEP activity itself does not lead to significant signs of an altered alcohol preference in mice but requires an environmental stimulus, our findings build a bridge between stress components and genetic factors in the development of alcoholism. Therefore, targeting NEP activity might be a very attractive approach for the treatment of alcohol abuse in a society with increasing social and financial stress.

New pathogenic thyrotropin receptor mutations decipher differentiated activity switching at a conserved helix 6 motif of family A GPCR
Biebermann(*), H., Winkler(*), F., Handke(*), D., Teichmann, A., Gerling(*), B., Cameron(*), F., Eichhorst, J., Grüters(*), A., Wiesner, B., Kühnen(*), P., Krude(*), H.; Kleinau(*), G.
The Journal of clinical endocrinology and metabolism, 97:E228-232

Tags: Cellular Imaging (Wiesner)

Abstract: CONTEXT: In this paper we report two new TSH receptor (TSHR) mutations. One mutation (Pro639(6.50)Leu) was identified in two siblings with congenital hypothyroidism, and a second mutation (Cys636(6.47)Arg) was found in a patient suffering from nonautoimmune hyperthyroidism. Both mutations are located in transmembrane helix (TMH) 6 at the conserved Cys(6.47)-Trp(Met)(6.48)-Leu(Ala)(6.49)-Pro(6.50) motif of family A G protein-coupled receptors (GPCR). OBJECTIVE: To study the pathogenic mechanisms, we tested patients' mutations and further side chain variations regarding their effects on TSHR signaling. RESULTS: Substitution Pro639Leu fully inactivates the promiscuous TSHR for cAMP (Gs) and IP (Gq) signaling. In contrast, Cys636Arg leads to constitutive activation of Gs. Organization of TSHR in oligomers was not modified by mutations at position 636. Interestingly, it is known from crystal structures of GPCR that Pro(6.50) is located at a TMH6 kink-distortion, which is a pivot during activation-related helical movements. However, the cell surface expressions of all mutants at position 639 were comparable to wild type, indicating a helical conformation like wild type. CONCLUSION: Until now, only naturally occurring constitutively activating mutations in TSHR TMH6 have been reported, but here we present the first pathogenic inactivating mutation (Pro639Leu). Our data are indicative of differentiated regulation of Gs and Gq signaling at particular TMH6 positions, but without any effects on TSHR oligomer constellation. Details of signaling modulation by each mutant at positions 636(6.47) and 639(6.50) help us to understand high conservation of these amino acids in family A GPCR. Described molecular (pathogenic) mechanisms are likely not unique for TSHR.

The proteoglycan syndecan 4 regulates transient receptor potential canonical 6 channels via RhoA/Rho-associated protein kinase signaling
Liu(*), Y., Echtermeyer(*), F., Thilo(*), F., Theilmeier(*), G., Schmidt, A., Schülein, R., Jensen(*), B. L., Loddenkemper(*), C., Jankowski(*), V., Marcussen(*), N., Gollasch(*), M., Arendshorst(*), W. J.; Tepel(*), M.
Arteriosclerosis, thrombosis, and vascular biology, 32:378-385

Tags: Protein Trafficking (Schülein)

Abstract: OBJECTIVE: Syndecan 4 (Sdc4) modulates signal transduction and regulates activity of protein channels. Sdc4 is essential for the regulation of cellular permeability. We hypothesized that Sdc4 may regulate transient receptor potential canonical 6 (TRPC6) channels, a determinant of glomerular permeability, in a RhoA/Rho-associated protein kinase-dependent manner. METHODS AND RESULTS: Sdc4 knockout (Sdc4(-/-)) mice showed increased glomerular filtration rate and ameliorated albuminuria under baseline conditions and after bovine serum albumin overload (each P<0.05). Using reverse transcription-polymerase chain reaction and immunoblotting, Sdc4(-/-) mice showed reduced TRPC6 mRNA by 79% and TRPC6 protein by 82% (each P<0.05). Sdc4(-/-) mice showed an increased RhoA activity by 87% and increased phosphorylation of ezrin in glomeruli by 48% (each P<0.05). Sdc4 knockdown in cultured podocytes reduced TRPC6 gene expression and reduced the association of TRPC6 with plasma membrane and TRPC6-mediated calcium influx and currents. Sdc4 knockdown inactivated negative regulatory protein Rho GTPase activating protein by 33%, accompanied by a 41% increase in RhoA activity and increased phosphorylation of ezrin (P<0.05). Conversely, overexpression of Sdc4 reduced RhoA activity and increased TRPC6 protein and TRPC6-mediated calcium influx and currents. CONCLUSIONS: Our results establish a previously unknown function of Sdc4 for regulation of TRPC6 channels and support the role of Sdc4 for the regulation of glomerular permeability.

Structural properties of EGCG-induced, nontoxic Alzheimer's disease Abeta oligomers
Lopez del Amo, J. M., Fink, U., Dasari, M., Grelle(*), G., Wanker(*), E. E., Bieschke(*), J.; Reif, B.
J Mol Biol, 421:517-524

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: The green tea compound epigallocatechin-3-gallate (EGCG) inhibits Alzheimer's disease beta-amyloid peptide (Abeta) neurotoxicity. Solution-state NMR allows probing initial EGCG-Abeta interactions. We show that EGCG-induced Abeta oligomers adopt a well-defined structure and are amenable for magic angle spinning solid-state NMR investigations. We find that EGCG interferes with the aromatic hydrophobic core of Abeta. The C-terminal part of the Abeta peptide (residues 22-39) adopts a beta-sheet conformation, whereas the N-terminus (residues 1-20) is unstructured. The characteristic salt bridge involving residues D23 and K28 is present in the structure of these oligomeric Abeta aggregates as well. The structural analysis of small-molecule-induced amyloid aggregates will open new perspectives for Alzheimer's disease drug development.

Saponins modulate the intracellular trafficking of protein toxins
Weng(*), A., Thakur(*), M., von Mallinckrodt(*), B., Beceren-Braun(*), F., Gilabert-Oriol(*), R., Wiesner, B., Eichhorst, J., Böttger(*), S., Melzig(*), M. F.; Fuchs(*), H.
J Control Release, 164:74-86

Tags: Cellular Imaging (Wiesner)

Abstract: Type I ribosome inactivating proteins such as saporin from the plant Saponaria officinalis L. are widely used as toxin moieties of targeted anti-tumor toxins. For exerting cytotoxicity the toxin moieties have to be released into the cytosol of tumor cells. However the cytosolic transfer of toxin molecules into the cytosol is mostly an inefficient process. In this report we demonstrate that certain saponins, which are also biosynthesized by Saponaria officinalis L., specifically mediate the release of saporin out of the intracellular compartments into the cytosol without affecting the integrity of the plasma membrane. The relevant cellular compartments were identified as late endosomes and lysosomes. Further studies revealed that endosomal acidification is a prerequisite for the saponin-mediated release of saporin. Binding analysis demonstrated an association of the saponins with saporin in a pH-dependent manner. The applicability of the saponin-mediated effect was demonstrated in vivo in a syngeneic tumor model using a saporin-based targeted anti-tumor toxin in combination with characterized saponins. (C) 2012 Elsevier B.V. All rights reserved.

Bioinorganic chemistry of copper coordination to alpha-synuclein: Relevance to Parkinson's disease
Binolfi, A., Quintanar(*), L., Bertoncini(*), C. W., Griesinger(*), C.; Fernandez(*), C. O.
Coordin Chem Rev, 256:2188-2201

Tags: In-Cell NMR (Selenko)

Abstract: Alpha-synuclein (AS) aggregation is associated with neurodegeneration in Parkinson's disease (PD). At the same time, alterations in metal ion homeostasis may play a pivotal role in the progression of AS amyloid assembly and the onset of PD. Elucidation of the structural basis directing AS-metal interactions and their effect on AS aggregation constitutes a key step toward understanding the role of metal ions in AS amyloid formation and neurodegeneration. This work provides a comprehensive review of recent advances attained in the bioinorganic chemistry of AS amyloid diseases. A hierarchy in AS-metal ion interactions has been established: while the physiologically relevant divalent metal ions iron and manganese interact at a non-specific, low-affinity binding interface in the C-terminus of AS, copper binds with high affinity at the N-terminal region and it is the most effective metal ion in accelerating AS filament assembly. The strong link between metal binding specificity and its impact on aggregation is discussed here on a mechanistic basis. A detailed description of the structural features and coordination environments of copper to AS is presented and discussed in the context of oxidative cellular events that might lead to the development of PD. Overall, the research observations presented here support the notion that perturbations in copper metabolism may be a common upstream event in the pathogenesis of neurodegenerative processes. (C) 2012 Elsevier B.V. All rights reserved.

Structural basis behind the interaction of Zn2+ with the protein alpha-synuclein and the A beta peptide: A comparative analysis
Valiente-Gabioud(*), A. A., Torres-Monserrat(*), V., Molina-Rubino(*), L., Binolfi, A., Griesinger(*), C.; Fernandez(*), C. O.
J Inorg Biochem, 117:334-341

Tags: In-Cell NMR (Selenko)

Abstract: alpha-Synuclein (AS) aggregation is associated to neurodegeneration in Parkinson's disease (PD). At the same time, alterations in metal ion homeostasis may play a pivotal role in the progression of AS amyloid assembly and the onset of PD. Elucidation of the structural basis directing AS metal interactions and their effect on AS aggregation constitutes a key step towards understanding the role of metal ions in AS amyloid formation and neurodegeneration. Despite of the reported evidences that link Zn2+ with the pathophysiology of PD and the fact that this metal ion was shown to promote AS fibrillation in vitro, neither the structural characterization of the binding sites nor the identification of the amino acids involved in the interaction of Zn2+ with the protein AS has been carried out. By using NMR spectroscopy, we have addressed here unknown structural details related to the binding of Zn2+ to the protein AS through the design of site-directed and domain truncated mutants of AS. The binding of zinc to the A beta peptide was also studied and discussed comparatively. Although the results of this study contribute to the understanding of the structural and molecular basis behind the acceleration of AS fibrillation mediated by Zn2+, the low affinity that characterizes the interaction of Zn2+ with AS contrasts strongly with the high-affinity features reported for the binding of this metal ion to other target proteins linked to human amylodosis such as A beta peptide and the Islet Amyloid Polypeptide (IAPP), challenging the biological relevance of zinc interactions in the pathogenesis of PD. (C) 2012 Elsevier Inc. All rights reserved.

Molecular Evolution of a Peptide GPCR Ligand Driven by Artificial Neural Networks
Bandholtz(*), S., Wichard, J., Kühne, R.; Grotzinger(*), C.
Plos One, 7

Tags: Computational Chemistry and Protein Design (Kühne)

Abstract: Peptide ligands of G protein-coupled receptors constitute valuable natural lead structures for the development of highly selective drugs and high-affinity tools to probe ligand-receptor interaction. Currently, pharmacological and metabolic modification of natural peptides involves either an iterative trial-and-error process based on structure-activity relationships or screening of peptide libraries that contain many structural variants of the native molecule. Here, we present a novel neural network architecture for the improvement of metabolic stability without loss of bioactivity. In this approach the peptide sequence determines the topology of the neural network and each cell corresponds one-to-one to a single amino acid of the peptide chain. Using a training set, the learning algorithm calculated weights for each cell. The resulting network calculated the fitness function in a genetic algorithm to explore the virtual space of all possible peptides. The network training was based on gradient descent techniques which rely on the efficient calculation of the gradient by back-propagation. After three consecutive cycles of sequence design by the neural network, peptide synthesis and bioassay this new approach yielded a ligand with 70fold higher metabolic stability compared to the wild type peptide without loss of the subnanomolar activity in the biological assay. Combining specialized neural networks with an exploration of the combinatorial amino acid sequence space by genetic algorithms represents a novel rational strategy for peptide design and optimization.

Functional Analysis of Centrosomal Kinase Substrates in Drosophila melanogaster Reveals a New Function of the Nuclear Envelope Component Otefin in Cell Cycle Progression
Habermann(*), K., Mirgorodskaya(*), E., Gobom(*), J., Lehmann(*), V., Müller(*), H., Blümlein(*), K., Deery(*), M. J., Czogiel(*), I., Erdmann, C., Ralser(*), M., von Kries, J. P.; Lange(*), B. M. H.
Mol Cell Biol, 32:3554-3569

Tags: Screening Unit (von Kries)

Abstract: Phosphorylation is one of the key mechanisms that regulate centrosome biogenesis, spindle assembly, and cell cycle progression. However, little is known about centrosome-specific phosphorylation sites and their functional relevance. Here, we identified phosphoproteins of intact Drosophila melanogaster centrosomes and found previously unknown phosphorylation sites in known and unexpected centrosomal components. We functionally characterized phosphoproteins and integrated them into regulatory signaling networks with the 3 important mitotic kinases, cdc2, polo, and aur, as well as the kinase CkII beta. Using a combinatorial RNA interference (RNAi) strategy, we demonstrated novel functions for P granule, nuclear envelope (NE), and nuclear proteins in centrosome duplication, maturation, and separation. Peptide microarrays confirmed phosphorylation of identified residues by centrosome-associated kinases. For a subset of phosphoproteins, we identified previously unknown centrosome and/or spindle localization via expression of tagged fusion proteins in Drosophila SL2 cells. Among those was otefin (Ote), an NE protein that we found to localize to centrosomes. Furthermore, we provide evidence that it is phosphorylated in vitro at threonine 63 (T63) through Aurora-A kinase. We propose that phosphorylation of this site plays a dual role in controlling mitotic exit when phosphorylated while dephosphorylation promotes G(2)/M transition in Drosophila SL2 cells.

GlialCAM, a Protein Defective in a Leukodystrophy, Serves as a CIC-2 Cl- Channel Auxiliary Subunit
Jeworutzki(*), E., Lopez-Hernandez(*), T., Capdevila-Nortes(*), X., Sirisi(*), S., Bengtsson, L., Montolio(*), M., Zifarelli(*), G., Arnedo(*), T., Müller(*), C. S., Schulte(*), U., Nunes(*), V., Martinez(*), A., Jentsch, T. J., Gasull(*), X., Pusch(*), M.; Estevez(*), R.
Neuron, 73:951-961

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Ion fluxes mediated by glial cells are required for several physiological processes such as fluid homeostasis or the maintenance of low extracellular potassium during high neuronal activity. In mice, the disruption of the Cl- channel CIC-2 causes fluid accumulation leading to myelin vacuolation. A similar vacuolation phenotype is detected in humans affected with megalencephalic leukoencephalopathy with subcortical cysts (MLC), a leukodystrophy which is caused by mutations in MLC1 or GLIALCAM. We here identify GlialCAM as a CIC-2 binding partner. GlialCAM and CIC-2 colocalize in Bergmann glia, in astrocyte-astrocyte junctions at astrocytic endfeet around blood vessels, and in myelinated fiber tracts. GlialCAM targets CIC-2 to cell junctions, increases CIC-2 mediated currents, and changes its functional properties. Disease-causing GLIALCAM mutations abolish the targeting of the channel to cell junctions. This work describes the first auxiliary subunit of CIC-2 and suggests that CIC-2 may play a role in the pathology of MLC disease.

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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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