FMP Publications

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

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References
The complex co-translational processing of glycoprotein GP5 of type 1 porcine reproductive and respiratory syndrome virus
Thaa(*), B., Kaufer(*), S., Neumann(*), S. A., Peibst(*), B., Nauwynck(*), H., Krause, E.; Veit(*), M.
Virus Res, 240:112-120
(2017)

Tags: Mass Spectrometry (Krause, E.)

Abstract: GP5 and M, the major membrane proteins of porcine reproductive and respiratory syndrome virus (PRRSV), are the driving force for virus budding and a target for antibodies. We studied co-translational processing of GP5 from an European PRRSV-1 strain. Using mass spectrometry, we show that in virus particles of a Lelystad variant, the signal peptide of GP5 was absent due to cleavage between glycine-34 and asparagine-35. This cleavage site removes an epitope for a neutralizing monoclonal antibody, but leaves intact another epitope recognized by neutralizing pig sera. Upon ectopic expression of this GP5 in cells, signal peptide cleavage was however inefficient. Complete cleavage occurred when cysteine-24 was changed to proline or an unused glycosylation site involving asparagine-35 was mutated. Insertion of proline at position 24 also caused carbohydrate attachment to asparagine-35. Glycosylation sites introduced downstream of residue 35 were used, but did not inhibit signal peptide processing. Co-expression of the M protein rescued this processing defect in GP5, suggesting a novel function of M towards GP5. We speculate that a complex interplay of the co-translational modifications of GP5 affect the N-terminal structure of the mature proteins and hence its antigenicity.

A lipid off-switch for mTORC1
Wallroth, A.; Haucke, V.
Mol Cell Oncol, 4:e1356899
(2017)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of metabolism, cell growth and survival. Our finding that local phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] synthesis at late endosomes/ lysosomes by class II PI3Kbeta (PI3KC2beta) represses mTORC1 identifies PI3KC2beta as a pharmacological target for the treatment of diabetes and cancer.

Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis
Walter(*), A. M., Müller(*), R., Tawfik(*), B., Wierda(*), K. D., Pinheiro(*), P. S., Nadler(*), A., McCarthy(*), A. W., Ziomkiewicz(*), I., Kruse(*), M., Reither(*), G., Rettig(*), J., Lehmann, M., Haucke, V., Hille(*), B., Schultz(*), C.; Sorensen(*), J. B.
Elife, 6
(2017)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging bypasses CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors.

Small-molecule inhibition of STOML3 oligomerization reverses pathological mechanical hypersensitivity
Wetzel(*), C., Pifferi(*), S., Picci(*), C., Gök(*), C., Hoffmann(*), D., Bali(*), K. K., Lampe, A., Lapatsina(*), L., Fleischer(*), R., Smith(*), E. S., Begay(*), V., Moroni(*), M., Estebanez(*), L., Kühnemund(*), J., Walcher(*), J., Specker, E., Neuenschwander, M., von Kries, J. P., Haucke, V., Kuner(*), R., Poulet(*), J. F., Schmoranzer(*), J., Poole(*), K.; Lewin(*), G. R.
Nat Neurosci, 20:209-218
(2017)

Tags: Molecular Pharmacology and Cell Biology (Haucke), Screening Unit (von Kries)

Abstract: The skin is equipped with specialized mechanoreceptors that allow the perception of the slightest brush. Indeed, some mechanoreceptors can detect even nanometer-scale movements. Movement is transformed into electrical signals via the gating of mechanically activated ion channels at sensory endings in the skin. The sensitivity of Piezo mechanically gated ion channels is controlled by stomatin-like protein-3 (STOML3), which is required for normal mechanoreceptor function. Here we identify small-molecule inhibitors of STOML3 oligomerization that reversibly reduce the sensitivity of mechanically gated currents in sensory neurons and silence mechanoreceptors in vivo. STOML3 inhibitors in the skin also reversibly attenuate fine touch perception in normal mice. Under pathophysiological conditions following nerve injury or diabetic neuropathy, the slightest touch can produce pain, and here STOML3 inhibitors can reverse mechanical hypersensitivity. Thus, small molecules applied locally to the skin can be used to modulate touch and may represent peripherally available drugs to treat tactile-driven pain following neuropathy.

GPCR-SSFE 2.0-a fragment-based molecular modeling web tool for Class A G-protein coupled receptors
Worth, C. L., Kreuchwig, F., Tiemann*, J. K. S., Kreuchwig, A., Ritschel*, M., Kleinau*, G., Hildebrand*, P. W.; Krause, G.
Nucleic Acids Res,
(2017)

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

Abstract: G-protein coupled receptors (GPCRs) are key players in signal transduction and therefore a large proportion of pharmaceutical drugs target these receptors. Structural data of GPCRs are sparse yet important for elucidating the molecular basis of GPCR-related diseases and for performing structure-based drug design. To ameliorate this problem, GPCR-SSFE 2.0 (http://www.ssfa-7tmr.de/ssfe2/), an intuitive web server dedicated to providing three-dimensional Class A GPCR homology models has been developed. The updated web server includes 27 inactive template structures and incorporates various new functionalities. Uniquely, it uses a fingerprint correlation scoring strategy for identifying the optimal templates, which we demonstrate captures structural features that sequence similarity alone is unable to do. Template selection is carried out separately for each helix, allowing both single-template models and fragment-based models to be built. Additionally, GPCR-SSFE 2.0 stores a comprehensive set of pre-calculated and downloadable homology models and also incorporates interactive loop modeling using the tool SL2, allowing knowledge-based input by the user to guide the selection process. For visual analysis, the NGL viewer is embedded into the result pages. Finally, blind-testing using two recently published structures shows that GPCR-SSFE 2.0 performs comparably or better than other state-of-the art GPCR modeling web servers.

A Two-Component Adhesive: Tau Fibrils Arise from a Combination of a Well-Defined Motif and Conformationally Flexible Interactions
Xiang(*), S. Q., Kulminskaya(*), N., Habenstein(*), B., Biernat(*), J., Tepper(*), K., Paulat(*), M., Griesinger(*), C., Becker(*), S., Lange, A., Mandelkow(*), E.; Linser(*), R.
J. Am. Chem. Soc., 139:2639-2646
(2017)

Tags: Molecular Biophysics (Lange, A.)

Abstract: Fibrillar aggregates of A beta and Tau in the brain are the major hallmarks of Alzheimer's disease. Most Tau fibers have a twisted appearance, but the twist can be variable and even absent. This ambiguity, which has also been associated with different phenotypes of tauopathies, has led to controversial assumptions about fibril constitution, and it is unclear to-date what the molecular causes of this polymorphism are. To tackle this question, we used solid-state NMR strategies providing assignments of non-seeded three-repeat-domain Tau(3RD) with an inherent heterogeneity. This is in contrast to the general approach to characterize the most homogeneous preparations by construct truncation or intricate seeding protocols. Here, carbon and nitrogen chemical-shift conservation between fibrils revealed invariable secondary-structure properties, however, with inter-monomer interactions variable among samples. Residues with variable amide shifts are localized mostly to N- and C-terminal regions within the rigid beta structure in the repeat region of Tau(3RD). By contrast, the hexapeptide motif in repeat R3, a crucial motif for fibril formation, shows strikingly low variability of all NMR parameters: Starting as a nucleation site for monomer monomer contacts, this six-residue sequence element also turns into a well-defined structural element upon fibril formation. Given the absence of external causes in vitro, the interplay of structurally differently conserved elements in this protein likely reflects an intrinsic property of Tau fibrils.

Unitary Properties of AMPA Receptors with Reduced Desensitization
Zhang(*), W., Eibl, C., Weeks(*), A. M., Riva, I., Li(*), Y. J., Plested, A. J. R.; Howe(*), J. R.
Biophys J,
(2017)

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: Wild-type AMPA receptors display a characteristic rapidly desensitizing phenotype. Many studies point to the dimer interface between pairs of extracellular ligand binding domains as the key region controlling the rate at which the receptors desensitize. However, mutations at the extracellular end of the pore-forming regions (near the putative ion channel gate) have also been shown to alter desensitization. Here we report the behavior of single GluA4 receptors carrying one of two mutations that greatly reduce desensitization at the level of ensemble currents: the dimer interface mutation L484Y and the Lurcher mutation (A623T, GluA4-Lc) in the extracellular end of M3 (the second true transmembrane helix). Analysis of unitary currents in patches with just one active receptor showed that each mutation greatly prolongs bursts of openings without prolonging the apparent duration of individual openings. Each mutation decreases the frequency with which individual receptors visit desensitized states, but both mutant receptors still desensitize multiple times per second. Cyclothiazide (CTZ) reduced desensitization of wild-type receptors and both types of mutant receptor. Analysis of shut-time distributions revealed a form of short-lived desensitization that was resistant to CTZ and was especially prominent for GluA4-Lc receptors. Despite reducing desensitization of GluA4 L484Y receptors, CTZ decreased the amplitude of ensemble currents through GluA2 and GluA4 LY receptor mutants. Single-channel analysis and comparison of the GluA2 L483Y ligand binding domain dimer in complex with glutamate with and without CTZ is consistent with the conclusion that CTZ binding to the dimer interface prevents effects of the LY mutation to modulate receptor activation, resulting in a reduction in the prevalence of large-conductance substates that accounts for the decrease in ensemble current amplitudes. Together, the results show that similar nondesensitizing AMPA-receptor phenotypes of population currents can arise from distinct underlying molecular mechanisms that produce different types of unitary activity.

Bacteriophage Tail-Tube Assembly Studied by Proton-Detected 4D Solid-State NMR
Zinke, M., Fricke, P., Samson(*), C., Hwang, S., Wall(*), J. S., Lange, S., Zinn-Justin(*), S.; Lange, A.
Angew Chem Int Ed Engl,
(2017)

Tags: Molecular Biophysics (Lange, A.)

Abstract: Obtaining unambiguous resonance assignments remains a major bottleneck in solid-state NMR studies of protein structure and dynamics. Particularly for supramolecular assemblies with large subunits (>150 residues), the analysis of crowded spectral data presents a challenge, even if three-dimensional (3D) spectra are used. Here, we present a proton-detected 4D solid-state NMR assignment procedure that is tailored for large assemblies. The key to recording 4D spectra with three indirect carbon or nitrogen dimensions with their inherently large chemical shift dispersion lies in the use of sparse non-uniform sampling (as low as 2 %). As a proof of principle, we acquired 4D (H)COCANH, (H)CACONH, and (H)CBCANH spectra of the 20 kDa bacteriophage tail-tube protein gp17.1 in a total time of two and a half weeks. These spectra were sufficient to obtain complete resonance assignments in a straightforward manner without use of previous solution NMR data.

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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
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