FMP Publications

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

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References
Small-molecule inhibitors of AF6 PDZ-mediated protein-protein interactions
Vargas, C., Radziwill(*), G., Krause, G., Diehl, A., Keller, S., Kamdem, N., Czekelius(*), C., Kreuchwig, A., Schmieder, P., Doyle(*), D., Moelling(*), K., Hagen, V., Schade(*), M.; Oschkinat, H.
Chemmedchem, 9:1458-1462
(2014)

Tags: NMR-Supported Structural Biology (Oschkinat), Solution NMR (Schmieder), Synthetic Organic Biochemistry (Hagen), Structural Bioinformatics and Protein Design (Krause, G.), Biophysics of Membrane Proteins (Keller), Synthetic Organic Biochemistry (Hagen)

Abstract: PDZ (PSD-95, Dlg, ZO-1) domains are ubiquitous interaction modules that are involved in many cellular signal transduction pathways. Interference with PDZ-mediated protein-protein interactions has important implications in disease-related signaling processes. For this reason, PDZ domains have gained attention as potential targets for inhibitor design and, in the long run, drug development. Herein we report the development of small molecules to probe the function of the PDZ domain from human AF6 (ALL1-fused gene from chromosome 6), which is an essential component of cell-cell junctions. These compounds bind to AF6 PDZ with substantially higher affinity than the peptide (Ile-Gln-Ser-Val-Glu-Val) derived from its natural ligand, EphB2. In intact cells, the compounds inhibit the AF6-Bcr interaction and interfere with epidermal growth factor (EGF)-dependent signaling.

AhR sensing of bacterial pigments regulates antibacterial defence
Moura-Alves(*), P., Fae(*), K., Houthuys(*), E., Dorhoi(*), A., Kreuchwig, A., Furkert, J., Barison(*), N., Diehl, A., Munder(*), A., Constant, P., Skrahina(*), T., Guhlich-Bornhof(*), U., Klemm(*), M., Koehler(*), A. B., Bandermann(*), S., Goosmann(*), C., Mollenkopf(*), H. J., Hurwitz(*), R., Brinkmann(*), V., Fillatreau(*), S., Daffe(*), M., Tummler, B., Kolbe(*), M., Oschkinat, H., Krause, G.; Kaufmann(*), S. H.
Nature, 512:387-392
(2014)

Tags: Structural Bioinformatics and Protein Design (Krause, G.), NMR-Supported Structural Biology (Oschkinat), Protein Trafficking (Schülein)

Abstract: The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR evolved to sense not only environmental pollutants but also microbial insults. We characterized bacterial pigmented virulence factors, namely the phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, as ligands of AhR. Upon ligand binding, AhR activation leads to virulence factor degradation and regulated cytokine and chemokine production. The relevance of AhR to host defence is underlined by heightened susceptibility of AhR-deficient mice to both P. aeruginosa and M. tuberculosis. Thus, we demonstrate that AhR senses distinct bacterial virulence factors and controls antibacterial responses, supporting a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigments as a new class of pathogen-associated molecular patterns.

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.

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
(2014)

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.

Quadruple-resonance magic-angle spinning NMR spectroscopy of deuterated solid proteins
Akbey, Ü., Nieuwkoop, A. J., Wegner, S., Voreck, A., Kunert, B., Bandara, P., Engelke, F., Nielsen, N. C.; Oschkinat, H.
Angew Chem Int Ed Engl, 53:2438-2442
(2014)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: (1)H-detected magic-angle spinning NMR experiments facilitate structural biology of solid proteins, which requires using deuterated proteins. However, often amide protons cannot be back-exchanged sufficiently, because of a possible lack of solvent exposure. For such systems, using (2)H excitation instead of (1)H excitation can be beneficial because of the larger abundance and shorter longitudinal relaxation time, T1, of deuterium. A new structure determination approach, "quadruple-resonance NMR spectroscopy", is presented which relies on an efficient (2)H-excitation and (2)H-(13)C cross-polarization (CP) step, combined with (1)H detection. We show that by using (2)H-excited experiments better sensitivity is possible on an SH3 sample recrystallized from 30 % H2O. For a membrane protein, the ABC transporter ArtMP in native lipid bilayers, different sets of signals can be observed from different initial polarization pathways, which can be evaluated further to extract structural properties.

Molecular characterization of ubiquitin-specific protease 18 reveals substrate specificity for interferon-stimulated gene 15
Basters(*), A., Geurink(*), P. P., El Oualid(*), F., Ketscher(*), L., Casutt(*), M. S., Krause, E., Ovaa(*), H., Knobeloch(*), K. P.; Fritz(*), G.
Febs J, 281:1918-1928
(2014)

Tags: Mass Spectrometry (Krause, E.)

Abstract: UNLABELLED: Protein modification by interferon-stimulated gene 15 (ISG15), an ubiquitin-like modifier, affects multiple cellular functions and represents one of the major antiviral effector systems. Covalent linkage of ISG15 to proteins was previously reported to be counteracted by ubiquitin-specific protease 18 (USP18). To date, analysis of the molecular properties of USP18 was hampered by low expression yields and impaired solubility. We established high-yield expression of USP18 in insect cells and purified the protease to homogeneity. USP18 binds with high affinity to ISG15, as shown by microscale thermophoresis with a Kd of 1.3 +/- 0.2 mum. The catalytic properties of USP18 were characterized by a novel assay using ISG15 fused to a fluorophore via an isopeptide bond, giving a Km of 4.6 +/- 0.2 mum and a kcat of 0.23 +/- 0.004 s(-1) , respectively, at pH 7.5. Furthermore, the recombinant enzyme cleaves efficiently ISG15 but not ubiquitin from endogenous cellular substrates. In line with these data, USP18 exhibited neither cross-reactivity with an ubiquitin isopeptide fluorophore substrate, nor with a ubiquitin vinyl sulfone, showing that the enzyme is specific for ISG15. STRUCTURED DIGITAL ABSTRACT: ISG15 and USP18 bind by microscale thermophoresis (View interaction) USP18 cleaves ISG15 by enzymatic study (View interaction).

[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)

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.

Cell penetrating peptides and cationic antibacterial peptides: two sides of the same coin
Rodriguez Plaza(*), J. G., Morales-Nava(*), R., Diener(*), C., Schreiber(*), G., Gonzalez(*), Z. D., Lara Ortiz(*), M. T., Ortega Blake(*), I., Pantoja(*), O., Volkmer, R., Klipp(*), E., Herrmann(*), A.; Del Rio(*), G.
J Biol Chem, 289:14448-14457
(2014)

Tags: Peptide Synthesis (Hackenberger/Volkmer)

Abstract: Cell penetrating peptides (CPP) and cationic antibacterial peptides (CAP) have similar physicochemical properties and yet it is not understood how such similar peptides display different activities. To address this question, we used Iztli peptide 1 (IP-1) because it has both CPP and CAP activities. Combining experimental and computational modeling of the internalization of IP-1, we show it is not internalized by receptor-mediated endocytosis, yet it permeates into many different cell types, including fungi and human cells. We also show that IP-1 makes pores in the presence of high electrical potential at the membrane, such as those found in bacteria and mitochondria. These results provide the basis to understand the functional redundancy of CPPs and CAPs.

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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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info(at)fmp-berlin.de

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