FMP Publications

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

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Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC
Voss, F. K., Ullrich, F., Münch, J., Lazarow, K., Lutter, D., Mah(*), N., Andrade-Navarro(*), M. A., von Kries, J. P., Stauber, T.; Jentsch, T. J.
Science, 344:634-638

Tags: Physiology and Pathology of Ion Transport (Jentsch), Screening Unit (von Kries)

Abstract: Regulation of cell volume is critical for many cellular and organismal functions, yet the molecular identity of a key player, the volume-regulated anion channel VRAC, has remained unknown. A genome-wide small interfering RNA screen in mammalian cells identified LRRC8A as a VRAC component. LRRC8A formed heteromers with other LRRC8 multispan membrane proteins. Genomic disruption of LRRC8A ablated VRAC currents. Cells with disruption of all five LRRC8 genes required LRRC8A cotransfection with other LRRC8 isoforms to reconstitute VRAC currents. The isoform combination determined VRAC inactivation kinetics. Taurine flux and regulatory volume decrease also depended on LRRC8 proteins. Our work shows that VRAC defines a class of anion channels, suggests that VRAC is identical to the volume-sensitive organic osmolyte/anion channel VSOAC, and explains the heterogeneity of native VRAC currents.

Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins
Wilhelm(*), B. G., Mandad(*), S., Truckenbrodt(*), S., Kröhnert(*), K., Schäfer(*), C., Rammner(*), B., Koo, S. J., Classen, G. A., Krauss, M., Haucke, V., Urlaub(*), H.; Rizzoli(*), S. O.
Science, 344:1023-1028

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Synaptic vesicle recycling has long served as a model for the general mechanisms of cellular trafficking. We used an integrative approach, combining quantitative immunoblotting and mass spectrometry to determine protein numbers; electron microscopy to measure organelle numbers, sizes, and positions; and super-resolution fluorescence microscopy to localize the proteins. Using these data, we generated a three-dimensional model of an "average" synapse, displaying 300,000 proteins in atomic detail. The copy numbers of proteins involved in the same step of synaptic vesicle recycling correlated closely. In contrast, copy numbers varied over more than three orders of magnitude between steps, from about 150 copies for the endosomal fusion proteins to more than 20,000 for the exocytotic ones.


Common gating of both CLC transporter subunits underlies voltage-dependent activation of the 2Cl-/1H+ exchanger ClC-7/Ostm1
Ludwig, C. F., Ullrich, F., Leisle, L., Stauber, T.; Jentsch, T. J.
J Biol Chem, 288:28611-28619

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: CLC anion transporters form dimers that function either as Cl(-) channels or as electrogenic Cl(-)/H(+) exchangers. CLC channels display two different types of "gates," "protopore" gates that open and close the two pores of a CLC dimer independently of each other and common gates that act on both pores simultaneously. ClC-7/Ostm1 is a lysosomal 2Cl(-)/1H(+) exchanger that is slowly activated by depolarization. This gating process is drastically accelerated by many CLCN7 mutations underlying human osteopetrosis. Making use of some of these mutants, we now investigate whether slow voltage activation of plasma membrane-targeted ClC-7/Ostm1 involves protopore or common gates. Voltage activation of wild-type ClC-7 subunits was accelerated by co-expressing an excess of ClC-7 subunits carrying an accelerating mutation together with a point mutation rendering these subunits transport-deficient. Conversely, voltage activation of a fast ClC-7 mutant could be slowed by co-expressing an excess of a transport-deficient mutant. These effects did not depend on whether the accelerating mutation localized to the transmembrane part or to cytoplasmic cystathionine-beta-synthase (CBS) domains of ClC-7. Combining accelerating mutations in the same subunit did not speed up gating further. No currents were observed when ClC-7 was truncated after the last intramembrane helix. Currents and slow gating were restored when the C terminus was co-expressed by itself or fused to the C terminus of the beta-subunit Ostm1. We conclude that common gating underlies the slow voltage activation of ClC-7. It depends on the CBS domain-containing C terminus that does not require covalent binding to the membrane domain of ClC-7.

NMR spectroscopy reveals unexpected structural variation at the protein-protein interface in MHC class I molecules
Beerbaum, M., Ballaschk, M., Erdmann, N., Schnick(*), C., Diehl, A., Uchanska-Ziegler(*), B., Ziegler(*), A.; Schmieder, P.
J Biomol NMR, 57:167-178

Tags: Solution NMR (Schmieder)

Abstract: beta2-Microglobulin (beta2m) is a small, monomorphic protein non-covalently bound to the heavy chain (HC) in polymorphic major histocompatibility complex (MHC) class I molecules. Given the high evolutionary conservation of structural features of beta2m in various MHC molecules as shown by X-ray crystallography, beta2m is often considered as a mere scaffolding protein. Using nuclear magnetic resonance (NMR) spectroscopy, we investigate here whether beta2m residues at the interface to the HC exhibit changes depending on HC polymorphisms and the peptides bound to the complex in solution. First we show that human beta2m can effectively be produced in deuterated form using high-cell-density-fermentation and we employ the NMR resonance assignments obtained for triple-labeled beta2m bound to the HLA-B*27:09 HC to examine the beta2m-HC interface. We then proceed to compare the resonances of beta2m in two minimally distinct subtypes, HLA-B*27:09 and HLA-B*27:05, that are differentially associated with the spondyloarthropathy Ankylosing Spondylitis. Each of these subtypes is complexed with four distinct peptides for which structural information is already available. We find that only the resonances at the beta2m-HC interface show a variation of their chemical shifts between the different complexes. This indicates the existence of an unexpected plasticity that enables beta2m to accommodate changes that depend on HC polymorphism as well as on the bound peptide through subtle structural variations of the protein-protein interface.

What's in a name? Why these proteins are intrinsically disordered: Why these proteins are intrinsically disordered
Dunker(*), A. K., Babu(*), M. M., Barbar(*), E., Blackledge(*), M., Bondos(*), S. E., Dosztanyi(*), Z., Dyson(*), H. J., Forman-Kay(*), J., Fuxreiter(*), M., Gsponer(*), J., Han(*), K. H., Jones(*), D. T., Longhi(*), S., Metallo(*), S. J., Nishikawa(*), K., Nussinov(*), R., Obradovic(*), Z., Pappu(*), R. V., Rost(*), B., Selenko, P., Subramaniam(*), V., Sussman(*), J. L., Tompa(*), P.; Uversky(*), V. N.
Intrinsically disordered proteins, 1:e24157

Tags: In-Cell NMR (Selenko)

Abstract: "What's in a name? That which we call a rose By any other name would smell as sweet." From "Romeo and Juliet", William Shakespeare (1594) This article opens a series of publications on disambiguation of the basic terms used in the field of intrinsically disordered proteins. We start from the beginning, namely from the explanation of what the expression "intrinsically disordered protein" actually means and why this particular term has been chosen as the common denominator for this class of proteins characterized by broad structural, dynamic and functional characteristics.

The alphabet of intrinsic disorder: I. Act like a Pro: On the abundance and roles of proline residues in intrinsically disordered proteins
Theillet, F. X., Kalmar(*), L., Tompa(*), P., Han(*), K. H., Selenko, P., Dunker(*), A. K., Daughdrill(*), G. W.; Uversky(*), V. N.
Intrinsically disordered proteins, 1:e24360

Tags: In-Cell NMR (Selenko)

Abstract: A significant fraction of every proteome is occupied by biologically active proteins that do not form unique three-dimensional structures. These intrinsically disordered proteins (IDPs) and IDP regions (IDPRs) have essential biological functions and are characterized by extensive structural plasticity. Such structural and functional behavior is encoded in the amino acid sequences of IDPs/IDPRs, which are enriched in disorder-promoting residues and depleted in order-promoting residues. In fact, amino acid residues can be arranged according to their disorder-promoting tendency to form an alphabet of intrinsic disorder that defines the structural complexity and diversity of IDPs/IDPRs. This review is the first in a series of publications dedicated to the roles that different amino acid residues play in defining the phenomenon of protein intrinsic disorder. We start with proline because data suggests that of the 20 common amino acid residues, this one is the most disorder-promoting.

A Floquet description of phase alternated sequences for efficient homonuclear recoupling in solid perdeuterated systems
Jayanthi(*), S., Akbey, Ü., Uluca(*), B., Oschkinat, H.; Vega(*), S.
Journal of Magnetic Resonance, 234:10-20

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: A Floquet description of a phase alternated homonuclear recoupling scheme for perdeuterated systems is presented. As a result, we demonstrate improvements in the recoupling efficiency of the DOuble Nucleus Enhanced Recoupling [DONER; J. Am. Chem. Soc. 131 (2009) 170541 technique by utilizing Phase Alternated Recoupling Irradiation Schemes [PARIS; Chem. Phys. Lett. 469 (2009) 342]. The effect of proton and deuterium radio frequency irradiation during recoupling has been systematically studied and theoretical observations have been verified experimentally using a deuterated model compound, L-Alanine, at 10 and 20 kHz magic angle spinning frequency. Experimental results are well in agreement with theoretical observations, thereby significantly increasing the recoupling efficiency of conventional DONER in perdeuterated systems. (C) 2013 Elsevier Inc. All rights reserved.

Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate
Posor, Y., Eichhorn-Grünig, M., Puchkov, D., Schöneberg(*), J., Ullrich(*), A., Lampe, A., Müller(*), R., Zarbakhsh(*), S., Gulluni(*), F., Hirsch(*), E., Krauss, M., Schultz(*), C., Schmoranzer, J., Noe(*), F.; Haucke, V.
Nature, 499:233-+

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Phosphoinositides serve crucial roles in cell physiology, ranging from cell signalling to membrane traffic(1,2). Among the seven eukaryotic phosphoinositides the best studied species is phosphatidylinositol-4,5-bisphosphate (PI(4,5)P-2), which is concentrated at the plasma membrane where, among other functions, it is required for the nucleation of endocytic clathrin-coated pits(3-6). No phosphatidylinositol other than PI(4,5)P-2 has been implicated in clathrin-mediated endocytosis, whereas the subsequent endosomal stages of the endocytic pathway are dominated by phosphatidylinositol-3-phosphates(PI(3)P)(7). How phosphatidylinositol conversion from PI(4,5)P-2-positive endocytic intermediates to PI(3)P-containing endosomes is achieved is unclear. Here we show that formation of phosphatidylinositol-3,4-bisphosphate (PI(3,4)P-2) by class II phosphatidylinositol-3-kinase C2 alpha (PI(3) K C2 alpha) spatiotemporally controls clathrin-mediated endocytosis. Depletion of PI(3,4)P-2 or PI(3)K C2 alpha impairs the maturation of late-stage clathrin-coated pits before fission. Timed formation of PI(3,4)P-2 by PI(3)K C2 alpha is required for selective enrichment of the BAR domain protein SNX9 at late-stage endocytic intermediates. These findings provide a mechanistic framework for the role of PI(3,4)P-2 in endocytosis and unravel a novel discrete function of PI(3,4)P-2 in a central cell physiological process.


A Single Glycine-Alanine Exchange Directs Ligand Specificity of the Elephant Progestin Receptor
Wierer(*), M., Schrey, A. K., Kühne, R., Ulbrich(*), S. E.; Meyer(*), H. H. D.
Plos One, 7

Tags: Computational Chemistry and Protein Design (Kühne)

Abstract: The primary gestagen of elephants is 5 alpha-dihydroprogesterone (DHP), which is unlike all other mammals studied until now. The level of DHP in elephants equals that of progesterone in other mammals, and elephants are able to bind DHP with similar affinity to progesterone indicating a unique ligand-binding specificity of the elephant progestin receptor (PR). Using site-directed mutagenesis in combination with in vitro binding studies we here report that this change in specificity is due to a single glycine to alanine exchange at position 722 (G722A) of PR, which specifically increases DHP affinity while not affecting binding of progesterone. By conducting molecular dynamics simulations comparing human and elephant PR ligand-binding domains (LBD), we observed that the alanine methyl group at position 722 is able to push the DHP A-ring into a position similar to progesterone. In the human PR, the DHP A-ring position is twisted towards helix 3 of PR thereby disturbing the hydrogen bond pattern around the C3-keto group, resulting in a lower binding affinity. Furthermore, we observed that the elephant PR ligand-binding pocket is more rigid than the human analogue, which probably explains the higher affinity towards both progesterone and DHP. Interestingly, the G722A substitution is not elephant-specific, rather it is also present in five independent lineages of mammalian evolution, suggesting a special role of the substitution for the development of distinct mammalian gestagen systems.


Generation and analyses of R8L barttin knockin mouse
Nomura(*), N., Tajima(*), M., Sugawara(*), N., Morimoto(*), T., Kondo(*), Y., Ohno(*), M., Uchida(*), K., Mutig(*), K., Bachmann(*), S., Soleimani(*), M., Ohta(*), E., Ohta(*), A., Sohara(*), E., Okado(*), T., Rai(*), T., Jentsch, T. J., Sasaki(*), S.; Uchida(*), S.
Am J Physiol-Renal, 301:F297-F307

Tags: Physiology and Pathology of Ion Transport (Jentsch

Abstract: Nomura N, Tajima M, Sugawara N, Morimoto T, Kondo Y, Ohno M, Uchida K, Mutig K, Bachmann S, Soleimani M, Ohta E, Ohta A, Sohara E, Okado T, Rai T, Jentsch TJ, Sasaki S, Uchida S. Generation and analyses of R8L barttin knockin mouse. Am J Physiol Renal Physiol 301: F297-F307, 2011. First published May 18, 2011; doi: 10.1152/ajprenal.00604.2010.-Barttin, a gene product of BSND, is one of four genes responsible for Bartter syndrome. Coexpression of barttin with ClC-K chloride channels dramatically induces the expression of ClC-K current via insertion of ClC-K-barttin complexes into plasma membranes. We previously showed that stably expressed R8L barttin, a disease-causing missense mutant, is retained in the endoplasmic reticulum (ER) of Madin-Darby canine kidney (MDCK) cells, with the barttin beta-subunit remaining bound to ClC-K alpha-subunits (Hayama A, Rai T, Sasaki S, Uchida S. Histochem Cell Biol 119: 485-493, 2003). However, transient expression of R8L barttin in MDCK cells was reported to impair ClC-K channel function without affecting its subcellular localization. To investigate the pathogenesis in vivo, we generated a knockin mouse model of Bartter syndrome that carries the R8L mutation. These mice display disease-like phenotypes (hypokalemia, metabolic alkalosis, and decreased NaCl reabsorption in distal tubules) under a low-salt diet. Immunofluorescence and immunoelectron microscopy revealed that the plasma membrane localization of both R8L barttin and the ClC-K channel was impaired in these mice, and transepithelial chloride transport in the thin ascending limb of Henle's loop (tAL) as well as thiazide-sensitive chloride clearance were significantly reduced. This reduction in transepithelial chloride transport in tAL, which is totally dependent on ClC-K1/barttin, correlated well with the reduction in the amount of R8L barttin localized to plasma membranes. These results suggest that the major cause of Bartter syndrome type IV caused by R8L barttin mutation is its aberrant intracellular localization.

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