FMP Publications

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

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References
Targeting G-protein-coupled receptors by Capture Compound Mass Spectrometry (CCMS) - a case study with sertindole
Blex(*), C., Michaelis(*), S., Schrey(*), A. K., Furkert, J., Eichhorst, J., Bartho(*), K., Quast(*), F. G., Marais(*), A., Hakelberg(*), M., Gruber(*), U., Niquet(*), S., Popp(*), O., Kroll(*), F., Sefkow(*), M., Schülein, R., Mathias(*), D.; Koster(*), H.
Chembiochem, 18:1639-1649
(2017)

Tags: Protein Trafficking (Schülein), Cellular Imaging (Wiesner/Puchkov)

Abstract: Unbiased chemoproteomic profiling of small molecule interactions with endogenous proteins is important for drug discovery. For meaningful results, all protein classes have to be tractable, including G-protein coupled receptors (GPCRs). These are hardly tractable by affinity pulldown from lysates. We report a Capture Compound (CC)-based strategy to target and identify GPCRs directly from living cells. We synthesized CCs with sertindole attached to the CC scaffold in different orientations to target the dopamine D2 receptor (DRD2) heterologously expressed in HEK293 cells. The structure-activity relationship of sertindole for DRD2 binding is reflected in the activities of the sertindole CCs in radioligand displacement, cell-based assays, and CCMS. The activity pattern was rationalized by molecular modelling. The most active CC showed activities very similar to unmodifed sertindole. Well below 100 fmol of DRD2 in living cells used as experiment input were sufficient for unambiguous identification of captured DRD2 by mass spectrometry. Our new CCMS workflow broadens the arsenal of chemoproteomic technologies to close a critical gap for the comprehensive characterization of drug-protein interactions.

Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders
Lorenz(*), C., Lesimple(*), P., Bukowiecki(*), R., Zink(*), A., Inak(*), G., Mlody(*), B., Singh(*), M., Semtner(*), M., Mah(*), N., Aure(*), K., Leong(*), M., Zabiegalov(*), O., Lyras(*), E. M., Pfiffer(*), V., Fauler(*), B., Eichhorst, J., Wiesner, B., Huebner(*), N., Priller(*), J., Mielke(*), T., Meierhofer(*), D., Izsvak(*), Z., Meier(*), J. C., Bouillaud(*), F., Adjaye(*), J., Schuelke(*), M., Wanker(*), E. E., Lombes(*), A.; Prigione(*), A.
Cell stem cell,
(2017)

Tags: Cellular Imaging (Wiesner)

Abstract: Mitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Modeling of these defects has been difficult because of the challenges associated with engineering mtDNA. We show here that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) retain the parental mtDNA profile and exhibit a metabolic switch toward oxidative phosphorylation. NPCs derived in this way from patients carrying a deleterious homoplasmic mutation in the mitochondrial gene MT-ATP6 (m.9185T>C) showed defective ATP production and abnormally high mitochondrial membrane potential (MMP), plus altered calcium homeostasis, which represents a potential cause of neural impairment. High-content screening of FDA-approved drugs using the MMP phenotype highlighted avanafil, which we found was able to partially rescue the calcium defect in patient NPCs and differentiated neurons. Overall, our results show that iPSC-derived NPCs provide an effective model for drug screening to target mtDNA disorders that affect the nervous system.

The ClC-K2 Chloride Channel Is Critical for Salt Handling in the Distal Nephron
Hennings(*), J. C., Andrini(*), O., Picard(*), N., Paulais(*), M., Huebner(*), A. K., Cayuqueo(*), I. K., Bignon(*), Y., Keck(*), M., Corniere(*), N., Böhm(*), D., Jentsch, T. J., Chambrey(*), R., Teulon(*), J., Hübner(*), C. A.; Eladari(*), D.
Journal of the American Society of Nephrology : JASN, 28:209-217
(2017)

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Chloride transport by the renal tubule is critical for blood pressure (BP), acid-base, and potassium homeostasis. Chloride uptake from the urinary fluid is mediated by various apical transporters, whereas basolateral chloride exit is thought to be mediated by ClC-Ka/K1 and ClC-Kb/K2, two chloride channels from the ClC family, or by KCl cotransporters from the SLC12 gene family. Nevertheless, the localization and role of ClC-K channels is not fully resolved. Because inactivating mutations in ClC-Kb/K2 cause Bartter syndrome, a disease that mimics the effects of the loop diuretic furosemide, ClC-Kb/K2 is assumed to have a critical role in salt handling by the thick ascending limb. To dissect the role of this channel in detail, we generated a mouse model with a targeted disruption of the murine ortholog ClC-K2. Mutant mice developed a Bartter syndrome phenotype, characterized by renal salt loss, marked hypokalemia, and metabolic alkalosis. Patch-clamp analysis of tubules isolated from knockout (KO) mice suggested that ClC-K2 is the main basolateral chloride channel in the thick ascending limb and in the aldosterone-sensitive distal nephron. Accordingly, ClC-K2 KO mice did not exhibit the natriuretic response to furosemide and exhibited a severely blunted response to thiazide. We conclude that ClC-Kb/K2 is critical for salt absorption not only by the thick ascending limb, but also by the distal convoluted tubule.

In colon epithelia, Clostridium perfringens enterotoxin causes focal leaks by targeting claudins which are apically accessible due to tight junction derangement
Eichner(*), M., Augustin(*), C., Fromm(*), A., Piontek, A., Walther(*), W., Bücker(*), R., Fromm(*), M., Krause, G., Schulzke(*), J. D., Günzel(*), D.; Piontek(*), J.
The Journal of infectious diseases,
(2017)

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

Abstract: Clostridium perfringens enterotoxin (CPE) causes food poisoning and antibiotic-associated diarrhea. It uses some claudin tight junction proteins (e.g. claudin-4) as receptors to form Ca2+-permeable pores in the membrane damaging epithelial cells in small intestine and colon. We demonstrate that only a subpopulation of colonic enterocytes which are characterized by apical dislocation of claudins are CPE-susceptible. CPE-mediated damage was enhanced if paracellular barrier was impaired by Ca2+-depletion, proinflammatory cytokine TNFalpha or dedifferentiation. Microscopy, Ca2+-monitoring, and electrophysiological data showed that CPE-mediated cytotoxicity and barrier disruption was limited by extent of CPE-binding. The latter was restricted by accessibility of non-junctional claudin molecules such as claudin-4 at apical membranes. Focal-leaks detected in HT-29/B6 colonic monolayers were verified for native tissue using colon biopsies. These mechanistic findings indicate how CPE-mediated effects may turn from self-limiting diarrhea into severe clinical manifestation such as colonic necrosis - if intestinal barrier dysfunction e.g. during inflammation facilitates claudin accessibility.

Targeting and alteration of tight junctions by bacteria and their virulence factors such as Clostridium perfringens enterotoxin
Eichner(*), M., Protze, J., Piontek, A., Krause, G.; Piontek(*), J.
Pflügers Arch, 469:77-90
(2017)

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

Abstract: The integrity of tight junctions, which regulate paracellular permeability, is challenged by many bacterial pathogens. This is caused by inflammatory responses triggered by pathogens and direct interaction of bacteria or their toxins with host epithelial cells. In some cases, tight junction proteins represent receptors for cell surface proteins or toxins of the pathogen, such as Clostridium perfringens enterotoxin (CPE). CPE causes diarrhea and cramps-the symptoms of a common foodborne illness, caused by C. perfringens type A. It uses a subgroup of the claudin family of tight junction proteins as receptors and forms pores in the membrane of intestinal epithelial cells. Ca2+ influx through these pores finally triggers cell damage. In this review, we summarize tight junction targeting and alteration by a multitude of different microorganisms such as C. perfringens, Escherichia coli, Helicobacter pylori, Salmonella typhimurium, Shigella flexneri, Vibrio cholerae, Yersinia enterocolitica, protozoan parasites, and their proteins. A focus is drawn towards CPE, the interaction with its receptors, cellular, and pathophysiological consequences for the intestinal epithelium. In addition, we portend to the use of CPE-based claudin modulators for drug delivery as well as diagnosis and therapy of cancer.

A cCPE-based xenon biosensor for magnetic resonance imaging of claudin-expressing cells
Piontek, A., Witte, C., May Rose, H., Eichner(*), M., Protze, J., Krause, G., Piontek(*), J.; Schröder, L.
Annals of the New York Academy of Sciences, 1397:195-208
(2017)

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

Abstract: The majority of malignant tumors originate from epithelial cells, and many of them are characterized by an overexpression of claudins (Cldns) and their mislocalization out of tight junctions. We utilized the C-terminal claudin-binding domain of Clostridium perfringens enterotoxin (cCPE), with its high affinity to specific members of the claudin family, as the targeting unit for a claudin-sensitive cancer biosensor. To overcome the poor sensitivity of conventional relaxivity-based magnetic resonance imaging (MRI) contrast agents, we utilized the superior sensitivity of xenon Hyper-CEST biosensors. We labeled cCPE for both xenon MRI and fluorescence detection. As one readout module, we employed a cryptophane (CrA) monoacid and, as the second, a fluorescein molecule. Both were conjugated separately to a biotin molecule via a polyethyleneglycol chemical spacer and later via avidin linked to GST-cCPE. Nontransfected HEK293 cells and HEK293 cells stably expressing Cldn4-FLAG were incubated with the cCPE-based biosensor. Fluorescence-based flow cytometry and xenon MRI demonstrated binding of the biosensor specifically to Cldn4-expressing cells. This study provides proof of concept for the use of cCPE as a carrier for diagnostic contrast agents, a novel approach for potential detection of Cldn3/-4-overexpressing tumors for noninvasive early cancer detection.

Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells
Appelt-Menzel(*), A., Cubukova(*), A., Günther(*), K., Edenhofer(*), F., Piontek(*), J., Krause, G., Stüber(*), T., Walles(*), H., Neuhaus(*), W.; Metzger(*), M.
Stem cell reports, 8:894-906
(2017)

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

Abstract: In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Omega cm2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies.

alpha-Synuclein lipoprotein nanoparticles
Eichmann, C., Bibow(*), S.; Riek(*), R.
Nanotechnol. Rev., 6:105-110
(2017)

Tags: In-Cell NMR (Selenko), NMR-Supported Structural Biology (Oschkinat)

Abstract: Apolipoprotein nanodiscs are a versatile tool in nanotechnology as membrane mimetics allowing, for example, the study of membrane proteins. It has recently been discovered that the Parkinson's disease associated protein alpha-synuclein (alpha-Syn) can also form discoid-like lipoprotein nanoparticles. The present review highlights the observation that a-Syn has the properties to define stable and homogeneous populations of nanoparticles with diameters of 7-10 nm and 19-28 nm by modifying lipid vesicles or encapsulating lipid bilayers in a nanodisctype fashion, respectively. In contrast to apolipoprotein nanodiscs, alpha-Syn nanoparticles can incorporate entirely negatively charged lipids emphasizing their potential use in nanotechnology as a negatively charged membrane mimetic.

High-density lipoprotein-like particle formation of Synuclein variants
Eichmann, C., Kumari(*), P.; Riek(*), R.
FEBS Lett, 591:304-311
(2017)

Tags: In-Cell NMR (Selenko)

Abstract: alpha-Synuclein (alpha-Syn) is an intrinsically disordered protein in solution whose fibrillar aggregates are the hallmark of Parkinson's disease (PD). Although the specific function of alpha-Syn is still unclear, its high structural plasticity is key for the interactions of alpha-Syn with biological membranes. Recently, it has been observed that alpha-Syn is able to form high-density lipoprotein-like (HDL-like) particles that are reminiscent of self-assembling phospholipid bilayer nanodiscs. Here, we extended our preparation method for the production of alpha-Syn lipoprotein particles to the beta- and gamma-Syn variants, and the PD-related familial alpha-Syn mutants. We show that all human Syns can form stable and homogeneous populations of HDL-like particles with distinct morphologies. Our results characterize the impact of the individual Syns on the formation capacity of these particles and indicate that Syn HDL-like particles are neither causing toxicity nor a toxicity-related loss of alpha-Syn in PD.

Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR
Erlendsson(*), S., Gotfryd(*), K., Larsen, F. H., Mortensen(*), J. S., Geiger, M. A., van Rossum, B. J., Oschkinat, H., Gether(*), U., Teilum(*), K.; Loland(*), C. J.
Elife, 6
(2017)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT. We were able to produce high-quality NMR spectra of substrate bound to microcrystalline LeuT samples and identify one set of sodium-dependent substrate-specific chemical shifts. Furthermore, our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein. We conclude that under our experimental conditions there is only one detectable leucine molecule bound to LeuT.

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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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