FMP Publications

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

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Don't flip out: AChRs are primed to catch and hold your attention
Plested, A. J.
Biophys J, 107:8-9

Tags: Molecular Neuroscience and Biophysics (Plested)

Gating characteristics control glutamate receptor distribution and trafficking in vivo
Petzoldt(*), A. G., Lee(*), Y. H., Khorramshahi(*), O., Reynolds(*), E., Plested, A. J., Herzel(*), H.; Sigrist(*), S. J.
Curr Biol, 24:2059-2065

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: Glutamate-releasing synapses dominate excitatory release in the brain. Mechanisms governing their assembly are of major importance for circuit development and long-term plasticity underlying learning and memory. AMPA/Kainate-type glutamate receptors (GluRs) are tetrameric ligand-gated ion channels that open their ion-conducting pores in response to binding of the neurotransmitter. Changes in subunit composition of postsynaptic GluRs are highly relevant for plasticity and development of glutamatergic synapses [1-4]. To date, posttranslational modifications, mostly operating via the intracellular C-terminal domains (CTDs) of GluRs, are presumed to be the major regulator of trafficking [5]. In recent years, structural and electrophysiological analyses have improved our understanding of GluR gating mechanism [6-11]. However, whether conformational changes subsequent to glutamate binding may per se be able to influence GluR trafficking has remained an unaddressed question. Using a Drosophila system allowing for extended visualization of GluR trafficking in vivo, we here provide evidence that mutations changing the gating behavior alter GluR distribution and trafficking. GluR mutants associated with reduced charge transfer segregated from coexpressed wild-type GluRs on the level of individual postsynaptic densities. Segregation was lost upon blocking of evoked glutamate release. Photobleaching experiments suggested increased mobility of mutants with reduced charge transfer, which accumulated prematurely during early steps of synapse assembly, but failed to further increase their level in accordance with assembly of the presynaptic scaffold. In summary, gating characteristics seem to be a new variable for the understanding of GluR trafficking relevant to both development and plasticity.

Photoinactivation of glutamate receptors by genetically encoded unnatural amino acids
Klippenstein, V., Ghisi, V., Wietstruk, M.; Plested, A. J.
J Neurosci, 34:980-991

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: Ionotropic glutamate receptors (iGluRs) are ubiquitous in the mammalian brain, and the AMPA-subtype is essential for fast, glutamate-activated postsynaptic currents. We incorporated photoactive crosslinkers into AMPA receptors using genetically encoded unnatural amino acid mutagenesis in a mammalian cell line. Receptors rescued by incorporation of unnatural amino acids, including p-benzoyl-l-phenylalanine (BzF, also known as Bpa), had largely similar properties to wild-type channels and were expressed at similar levels. BzF incorporation at subunit interfaces afforded photocrosslinking of subunits, as assessed by biochemical experiments. In electrophysiological recordings, BzF incorporation allowed selective and potent UV-driven photoinactivation of both homomeric (GluA2) and heteromeric (GluA2:GluA1) AMPA receptors. State dependence of trapping at two sites in the lower lobe of the ligand binding domain is consistent with deformation of these domains as well as intersubunit rearrangements during AMPA receptor desensitization.

Mechanism of Modulation of AMPA Receptors by Stargazin
Carbone, A. L.; Plested, A. J.
Biophys. J., 106:150a-150a

Tags: Molecular Neuroscience and Biophysics (Plested)

Claudin-3 and claudin-5 protein folding and assembly into the tight junction are controlled by non-conserved residues in the transmembrane 3 (TM3) and extracellular loop 2 (ECL2) segments
Rossa, J., Ploeger, C., Vorreiter, F., Saleh, T., Protze, J., Günzel, D., Wolburg, H., Krause, G.; Piontek, J.
J Biol Chem, 289:7641-7653

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

Abstract: The mechanism of tight junction (TJ) assembly and the structure of claudins (Cldn) that form the TJ strands are unclear. This limits the molecular understanding of paracellular barriers and strategies for drug delivery across tissue barriers. Cldn3 and Cldn5 are both common in the blood-brain barrier but form TJ strands with different ultrastructures. To identify the molecular determinants of folding and assembly of these classic claudins, Cldn3/Cldn5 chimeric mutants were generated and analyzed by cellular reconstitution of TJ strands, live cell confocal imaging, and freeze-fracture electron microscopy. A comprehensive screening was performed on the basis of the rescue of mutants deficient for strand formation. Cldn3/Cldn5 residues in transmembrane segment 3, TM3 (Ala-127/Cys-128, Ser-136/Cys-137, Ser-138/Phe-139), and the transition of TM3 to extracellular loop 2, ECL2 (Thr-141/Ile-142) and ECL2 (Asn-148/Asp-149, Leu-150/Thr-151, Arg-157/Tyr-158), were identified to be involved in claudin folding and/or assembly. Blue native PAGE and FRET assays revealed 1% n-dodecyl beta-d-maltoside-resistant cis-dimerization for Cldn5 but not for Cldn3. This homophilic interaction was found to be stabilized by residues in TM3. The resulting subtype-specific cis-dimer is suggested to be a subunit of polymeric TJ strands and contributes to the specific ultrastructure of the TJ detected by freeze-fracture electron microscopy. In particular, the Cldn5-like exoplasmic face-associated and particle-type strands were found to be related to cis-dimerization. These results provide new insight into the mechanisms of paracellular barrier formation by demonstrating that defined non-conserved residues in TM3 and ECL2 of classic claudins contribute to the formation of TJ strands with differing ultrastructures.

Molecular and structural transmembrane determinants critical for embedding claudin-5 into tight junctions reveal a distinct four-helix bundle arrangement
Rossa, J., Protze, J., Kern, C., Piontek, A., Günzel(*), D., Krause, G.; Piontek(*), J.
Biochem J, 464:49-60

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

Abstract: The mechanism of TJ (tight junction) assembly and the structure of TJ strand-forming Cldns (claudins) are unclear. To identify determinants of assembly of blood-brain barrier-related Cldn3 and Cldn5, chimaeric mutants were analysed by cellular reconstitution of TJ strands and live-cell imaging. On the basis of the rescue of mutants deficient for strand formation, we identified Cldn5 residues (Cys128, Ala132, Ile142, Ala163, Ile166 and Leu174) involved in Cldn folding and assembly. Experimental results were combined with structural bioinformatics approaches. Initially the experimentally validated previous model of the ECL2 (extracellular loop 2) of Cldn5 was extended to the flanking transmembrane segments (TM3/TM4). A coiled-coil interface probably caused by alternating small and large residues is supported by concomitant knob-into-hole interactions including Cldn5-specific residues identified in the present paper. To address arrangement of the TMs in a four-helix bundle, data from evolutionary sequence couplings and comparative modelling of intramolecular interfaces in the transmembrane region of Cldns led to a complete Cldn5 model. Our suggested Cldn subtype-specific intramolecular interfaces that are formed by conserved coiled-coil motifs and non-conserved residues in distinct TM positions were confirmed by the recently released crystal structure of Cldn15. The identified molecular and structural determinants essentially contribute to assembly of Cldns into TJ strands.

Activation of Ligand Binding Domains of an AMPA-Type Glutamate Receptor
Baranovic, J., Chebli, M., Salazar, H. P., Faelber(*), K., Ghisi, V., Lau(*), A. Y., Daumke(*), O.; Plested, A. J. R.
Biophys. J., 106:29a-29a

Tags: Molecular Neuroscience and Biophysics (Plested)

Rapid proton-detected NMR assignment for proteins with fast magic angle spinning
Barbet-Massin(*), E., Pell(*), A. J., Retel, J. S., Andreas(*), L. B., Jaudzems(*), K., Franks, W. T., Nieuwkoop, A. J., Hiller, M., Higman(*), V., Guerry(*), P., Bertarello(*), A., Knight(*), M. J., Felletti(*), M., Le Marchand(*), T., Kotelovica(*), S., Akopjana(*), I., Tars(*), K., Stoppini(*), M., Bellotti(*), V., Bolognesi(*), M., Ricagno(*), S., Chou(*), J. J., Griffin(*), R. G., Oschkinat, H., Lesage(*), A., Emsley(*), L., Herrmann(*), T.; Pintacuda(*), G.
J Am Chem Soc, 136:12489-12497

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Using a set of six (1)H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5-30 kDa proteins. The approach relies on perdeuteration, amide (2)H/(1)H exchange, high magnetic fields, and high-spinning frequencies (omegar/2pi >/= 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary (13)C/(15)N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.

CLCN7 and TCIRG1 Mutations Differentially Affect Bone Matrix Mineralization in Osteopetrotic Individuals
Barvencik(*), F., Kurth(*), I., Koehne(*), T., Stauber, T., Zustin(*), J., Tsiakas, K., Ludwig, C. F., Beil(*), F. T., Pestka(*), J. M., Hahn(*), M., Santer(*), R., Supanchart(*), C., Kornak(*9, U., Del Fattore(*), A., Jentsch, T. J., Teti(*), A., Schulz(*), A., Schinke(*), T.; Amling(*), M.
J Bone Miner Res, 29:982-991

Tags: Physiology and Pathology of Ion Transport (Jentsch)

N-[6-(4-butanoyl-5-methyl-1H-pyrazol-1-yl)pyridazin-3-yl]-5-chloro-1-[2-(4-methyl piperazin-1-yl)-2-oxoethyl]-1H-indole-3-carboxamide (SAR216471), a novel intravenous and oral, reversible, and directly acting P2Y12 antagonist
Boldron(*), C., Besse(*), A., Bordes(*), M. F., Tissandie(*), S., Yvon(*), X., Gau(*), B., Badorc(*), A., Rousseaux(*), T., Barre(*), G., Meneyrol(*), J., Zech(*), G., Nazare, M., Fossey(*), V., Pflieger(*), A. M., Bonnet-Lignon(*), S., Millet(*), L., Briot(*), C., Dol(*), F., Herault(*), J. P., Savi(*), P., Lassalle(*), G., Delesque(*), N., Herbert(*), J. M.; Bono(*), F.
Journal of medicinal chemistry, 57:7293-7316

Tags: Medicinal Chemistry (Nazare)

Abstract: In the search of a potential backup for clopidogrel, we have initiated a HTS campaign designed to identify novel reversible P2Y12 antagonists. Starting from a hit with low micromolar binding activity, we report here the main steps of the optimization process leading to the identification of the preclinical candidate SAR216471. It is a potent, highly selective, and reversible P2Y12 receptor antagonist and by far the most potent inhibitor of ADP-induced platelet aggregation among the P2Y12 antagonists described in the literature. SAR216471 displays potent in vivo antiplatelet and antithrombotic activities and has the potential to differentiate from other antiplatelet agents.

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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
+4930 94793 - 100 
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