FMP Publications

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

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Flexible, polymer-supported synthesis of sphingosine derivatives provides ceramides with enhanced biological activity
El-Dahshan, A., Al-Gharabli(*), S. I., Radetzki, S., Al-Tel(*), T. H., Kumar(*), P.; Rademann, J.
Bioorg Med Chem, 22:5506-5512

Tags: Medicinal Chemistry (Rademann), Screening Unit (von Kries)

Abstract: A polymer-supported route for the synthesis of sphingosine derivatives is presented based on the C-acylation of polymeric phosphoranylidene acetates with an Fmoc-protected amino acid. The approach enables the flexible variation of the sphingosine tail through a deprotection-decarboxylation sequence followed by E-selective Wittig olefination cleavage. d-Erythro-sphingosine analogs have been synthesized by diastereoselective reduction of the keto group employing LiAlH(O-tBu)3 as reducing agent. The effect of ceramides and keto-ceramides on the proliferation of three cancer cell lines HEP G-2, PC-12 and HL-60 was investigated and a ceramide containing an aromatic sphingosine tail was identified as being most active.

Reporter assay for endo/lysosomal escape of toxin-based therapeutics
Gilabert-Oriol(*), R., Thakur(*), M., von Mallinckrodt(*), B., Bhargava(*), C., Wiesner, B., Eichhorst, J., Melzig(*), M. F., Fuchs(*), H.; Weng(*), A.
Toxins (Basel), 6:1644-1666

Tags: Cellular Imaging (Wiesner)

Abstract: Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters-horseradish peroxidase (HRP), Alexa Fluor 488 (Alexa) and ricin A-chain (RTA)-were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates-saporin-HRP, (Alexa)saporin and saporin-KQ-RTA-were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of (Alexa)saporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10-1000 nM.

Proteome analysis of the HIV-1 Gag interactome
Engeland(*), C. E., Brown(*), N. P., Borner(*), K., Schümann, M., Krause, E., Kaderali(*), L., Müller(*), G. A.; Kräusslich(*), H. G.
Virology, 460-461:194-206

Tags: Mass Spectrometry (Krause, E.)

Abstract: Human immunodeficiency virus Gag drives assembly of virions in infected cells and interacts with host factors which facilitate or restrict viral replication. Although several Gag-binding proteins have been characterized, understanding of virus-host interactions remains incomplete. In a series of six affinity purification screens, we have identified protein candidates for interaction with HIV-1 Gag. Proteins previously found in virions or identified in siRNA screens for host factors influencing HIV-1 replication were recovered. Helicases, translation factors, cytoskeletal and motor proteins, factors involved in RNA degradation and RNA interference were enriched in the interaction data. Cellular networks of cytoskeleton, SR proteins and tRNA synthetases were identified. Most prominently, components of cytoplasmic RNA transport granules were co-purified with Gag. This study provides a survey of known Gag-host interactions and identifies novel Gag binding candidates. These factors are associated with distinct molecular functions and cellular pathways relevant in host-pathogen interactions.

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.

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

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

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).

GlialCAM, a CLC-2 Cl(-) channel subunit, activates the slow gate of CLC chloride channels
Jeworutzki(*), E., Lagostena(*), L., Elorza-Vidal(*), X., Lopez-Hernandez, T., Estevez(*), R.; Pusch(*), M.
Biophys J, 107:1105-1116

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: GlialCAM, a glial cell adhesion molecule mutated in megalencephalic leukoencephalopathy with subcortical cysts, targets the CLC-2 Cl(-) channel to cell contacts in glia and activates CLC-2 currents in vitro and in vivo. We found that GlialCAM clusters all CLC channels at cell contacts in vitro and thus studied GlialCAM interaction with CLC channels to investigate the mechanism of functional activation. GlialCAM slowed deactivation kinetics of CLC-Ka/barttin channels and increased CLC-0 currents opening the common gate and slowing its deactivation. No functional effect was seen for common gate deficient CLC-0 mutants. Similarly, GlialCAM targets the common gate deficient CLC-2 mutant E211V/H816A to cell contacts, without altering its function. Thus, GlialCAM is able to interact with all CLC channels tested, targeting them to cell junctions and activating them by stabilizing the open configuration of the common gate. These results are important to better understand the physiological role of GlialCAM/CLC-2 interaction.

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

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.

Design of a General-Purpose European Compound Screening Library for EU-OPENSCREEN
Horvath(*), D., Lisurek, M., Rupp, B., Kühne, R., Specker, E., von Kries, J., Rognan(*), D., Andersson(*), C. D., Almqvist(*), F., Elofsson(*), M., Enqvist(*), P. A., Gustavsson(*), A. L., Remez(*), N., Mestres(*), J., Marcou(*), G., Varnek(*), A., Hibert(*), M., Quintana(*), J.; Frank, R.
Chemmedchem, 9:2309-2326

Tags: Chemical Systems Biology (Frank), Screening Unit (von Kries), Computational Chemistry and Protein Design (Kühne)

Abstract: This work describes a collaborative effort to define and apply a protocol for the rational selection of a general-purpose screening library, to be used by the screening platforms affiliated with the EU-OPENSCREEN initiative. It is designed as a standard source of compounds for primary screening against novel biological targets, at the request of research partners. Given the general nature of the potential applications of this compound collection, the focus of the selection strategy lies on ensuring chemical stability, absence of reactive compounds, screening-compliant physicochemical properties, loose compliance to drug-likeness criteria (as drug design is a major, but not exclusive application), and maximal diversity/coverage of chemical space, aimed at providing hits for a wide spectrum of drugable targets. Finally, practical availability/cost issues cannot be avoided. The main goal of this publication is to inform potential future users of this library about its conception, sources, and characteristics. The outline of the selection procedure, notably of the filtering rules designed by a large committee of European medicinal chemists and chemoinformaticians, may be of general methodological interest for the screening/medicinal chemistry community. The selection task of 200K molecules out of a pre-filtered set of 1.4M candidates was shared by five independent European research groups, each picking a subset of 40K compounds according to their own in-house methodology and expertise. An in-depth analysis of chemical space coverage of the library serves not only to characterize the collection, but also to compare the various chemoinformatics-driven selection procedures of maximal diversity sets. Compound selections contributed by various participating groups were mapped onto general-purpose self-organizing maps (SOMs) built on the basis of marketed drugs and bioactive reference molecules. In this way, the occupancy of chemical space by the EU-OPENSCREEN library could be directly compared with distributions of known bioactives of various classes. This mapping highlights the relevance of the selection and shows how the consensus reached by merging the five different 40K selections contributes to achieve this relevance. The approach also allows one to readily identify subsets of target-or target-class-oriented compounds from the EU-OPENSCREEN library to suit the needs of the diverse range of potential users. The final EU-OPENSCREEN library, assembled by merging five independent selections of 40K compounds from various expert groups, represents an excellent example of a Europe-wide collaborative effort toward the common objective of building best-in-class European open screening platforms.

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

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.

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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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