FMP Publications

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

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References
An air-liquid interphase approach for modeling the early embryo-maternal contact zone
Chen(*), S., Palma-Vera(*), S. E., Langhammer(*), M., Galuska(*), S. P., Braun(*), B. C., Krause, E., Lucas-Hahn(*), A.; Schoen(*), J.
Sci Rep, 7:42298
(2017)

Tags: Mass Spectrometry (Krause, E.)

Abstract: We developed an air-liquid interphase culture procedure for mammalian oviduct epithelial cells leading to the formation of functional epithelial tissues, which generate oviduct fluid surrogates. These in vitro oviduct epithelia can be co-cultured with living zygotes and enable embryonic development up to the blastocyst stage without addition of embryo culture medium. The described strategy is broadly applicable to analyze early embryo-maternal interactions under standardized in vitro conditions.

Structural Characterization and Ligand/Inhibitor Identification Provide Functional Insights into the Mycobacterium tuberculosis Cytochrome P450 CYP126A1
Chenge(*), J. T., Duyet(*), L. V., Swami(*), S., McLean(*), K. J., Kavanagh(*), M. E., Coyne(*), A. G., Rigby(*), S. E., Cheesman(*), M. R., Girvan(*), H. M., Levy(*), C. W., Rupp, B., von Kries, J. P., Abell(*), C., Leys(*), D.; Munro(*), A. W.
J Biol Chem, 292:1310-1329
(2017)

Tags: Screening Unit (von Kries), Computational Chemistry and Protein Design (Kühne)

Abstract: The Mycobacterium tuberculosis H37Rv genome encodes 20 cytochromes P450, including P450s crucial to infection and bacterial viability. Many M. tuberculosis P450s remain uncharacterized, suggesting that their further analysis may provide new insights into M. tuberculosis metabolic processes and new targets for drug discovery. CYP126A1 is representative of a P450 family widely distributed in mycobacteria and other bacteria. Here we explore the biochemical and structural properties of CYP126A1, including its interactions with new chemical ligands. A survey of azole antifungal drugs showed that CYP126A1 is inhibited strongly by azoles containing an imidazole ring but not by those tested containing a triazole ring. To further explore the molecular preferences of CYP126A1 and search for probes of enzyme function, we conducted a high throughput screen. Compounds containing three or more ring structures dominated the screening hits, including nitroaromatic compounds that induce substrate-like shifts in the heme spectrum of CYP126A1. Spectroelectrochemical measurements revealed a 155-mV increase in heme iron potential when bound to one of the newly identified nitroaromatic drugs. CYP126A1 dimers were observed in crystal structures of ligand-free CYP126A1 and for CYP126A1 bound to compounds discovered in the screen. However, ketoconazole binds in an orientation that disrupts the BC-loop regions at the P450 dimer interface and results in a CYP126A1 monomeric crystal form. Structural data also reveal that nitroaromatic ligands "moonlight" as substrates by displacing the CYP126A1 distal water but inhibit enzyme activity. The relatively polar active site of CYP126A1 distinguishes it from its most closely related sterol-binding P450s in M. tuberculosis, suggesting that further investigations will reveal its diverse substrate selectivity.

Measurement of backbone hydrogen-deuterium exchange in the type III secretion system needle protein PrgI by solid-state NMR
Chevelkov, V., Giller(*), K., Becker(*), S.; Lange, A.
J Magn Reson, 283:110-116
(2017)

Tags: Molecular Biophysics (Lange, A.)

Abstract: In this report we present site-specific measurements of amide hydrogen-deuterium exchange rates in a protein in the solid state phase by MAS NMR. Employing perdeuteration, proton detection and a high external magnetic field we could adopt the highly efficient Relax-EXSY protocol previously developed for liquid state NMR. According to this method, we measured the contribution of hydrogen exchange on apparent 15N longitudinal relaxation rates in samples with differing D2O buffer content. Differences in the apparent T1 times allowed us to derive exchange rates for multiple residues in the type III secretion system needle protein.

Trictide, a tricellulin-derived peptide to overcome cellular barriers
Cording, J., Arslan, B., Staat, C., Dithmer, S., Krug(*), S. M., Krüger(*), A., Berndt, P., Günther, R., Winkler, L., Blasig, I. E.; Haseloff, R. F.
Annals of the New York Academy of Sciences,
(2017)

Tags: Molecular Cell Physiology (Blasig, I.E.)

Abstract: The majority of tight junction (TJ) proteins restrict the paracellular permeation of solutes via their extracellular loops (ECLs). Tricellulin tightens tricellular TJs (tTJs) and regulates bicellular TJ (bTJ) proteins. We demonstrate that the addition of recombinantly produced extracellular loop 2 (ECL2) of tricellulin opens cellular barriers. The peptidomimetic trictide, a synthetic peptide derived from tricellulin ECL2, increases the passage of ions, as well as of small and larger molecules up to 10 kDa, between 16 and 30 h after application to human epithelial colorectal adenocarcinoma cell line 2. Tricellulin and lipolysis-stimulated lipoprotein receptor relocate from tTJs toward bTJs, while the TJ proteins claudin-1 and occludin redistribute from bTJs to the cytosol. Analyzing the opening of the tricellular sealing tube by the peptidomimetic using super-resolution stimulated-emission depletion microscopy revealed a tricellulin-free area at the tricellular region. Cis-interactions (as measured by fluorescence resonance energy transfer) of tricellulin-tricellulin (tTJs), tricellulin-claudin-1, tricellulin-marvelD3, and occludin-occludin (bTJs) were strongly affected by trictide treatment. Circular dichroism spectroscopy and molecular modeling suggest that trictide adopts a beta-sheet structure, resulting in a peculiar interaction surface for its binding to tricellulin. In conclusion, trictide is a novel and promising tool for overcoming cellular barriers at bTJs and tTJs with the potential to transiently improve drug delivery.

Claudin peptidomimetics modulate tissue barriers for enhanced drug delivery
Dithmer, S., Staat, C., Müller, C., Ku(*), M. C., Pohlmann(*), A., Niendorf(*), T., Gehne, N., Fallier-Becker(*), P., Kittel(*), A., Walter(*), F. R., Veszelka(*), S., Deli(*), M. A., Blasig, R., Haseloff, R. F., Blasig, I. E.; Winkler, L.
Annals of the New York Academy of Sciences, 1397:169-184
(2017)

Tags: Molecular Cell Physiology (Blasig, I.E.)

Abstract: The blood-brain barrier (BBB) formed by the microvascular endothelium limits cerebral drug delivery. The paraendothelial cleft is sealed by tight junctions (TJs) with a major contribution from claudin-5, which we selected as a target to modulate BBB permeability. For this purpose, drug-enhancer peptides were designed based on the first extracellular loop (ECL) of claudin-5 to allow transient BBB permeabilization. Peptidomimetics (C5C2 and derivatives, nanomolar affinity to claudin-5) size-selectively (</=40 kDa) and reversibly (12-48 h) increased the permeability of brain endothelial and claudin-5-transfected epithelial cell monolayers. Upon peptide uptake, the number of TJ strand particles diminished, claudin-5 was downregulated and redistributed from cell-cell contacts to the cytosol, and the cell shape was altered. Cellular permeability of doxorubicin (cytostatic drug, 580 Da) was enhanced after peptide administration. Mouse studies (3.5 mumol/kg i.v.) confirmed that, for both C5C2 and a d-amino acid derivative, brain uptake of Gd-diethylene-triamine penta-acetic acid (547 Da) was enhanced within 4 h of treatment. On the basis of our functional data, circular dichroism measurements, molecular modeling, and docking experiments, we suggest an association model between beta-sheets flanked by alpha-helices, formed by claudin-5 ECLs, and the peptides. In conclusion, we identified claudin-5 peptidomimetics that improve drug delivery through endothelial and epithelial barriers expressing claudin-5.

Pharmacological restoration and therapeutic targeting of the B-cell phenotype in classical Hodgkin lymphoma
Du(*), J., Neuenschwander, M., Yu(*), Y., Dabritz(*), J. H., Neuendorff(*), N. R., Schleich(*), K., Bittner(*), A., Milanovic(*), M., Beuster(*), G., Radetzki, S., Specker, E., Reimann(*), M., Rosenbauer(*), F., Mathas(*), S., Lohneis(*), P., Hummel(*), M., Dörken(*), B., von Kries, J. P., Lee(*), S.; Schmitt(*), C. A.
Blood, 129:71-81
(2017)

Tags: Screening Unit (von Kries)

Abstract: Classical Hodgkin lymphoma (cHL), although originating from B cells, is characterized by the virtual lack of gene products whose expression constitutes the B-cell phenotype. Epigenetic repression of B-cell-specific genes via promoter hypermethylation and histone deacetylation as well as compromised expression of B-cell-committed transcription factors were previously reported to contribute to the lost B-cell phenotype in cHL. Restoring the B-cell phenotype may not only correct a central malignant property, but it may also render cHL susceptible to clinically established antibody therapies targeting B-cell surface receptors or small compounds interfering with B-cell receptor signaling. We conducted a high-throughput pharmacological screening based on >28 000 compounds in cHL cell lines carrying a CD19 reporter to identify drugs that promote reexpression of the B-cell phenotype. Three chemicals were retrieved that robustly enhanced CD19 transcription. Subsequent chromatin immunoprecipitation-based analyses indicated that action of 2 of these compounds was associated with lowered levels of the transcriptionally repressive lysine 9-trimethylated histone H3 mark at the CD19 promoter. Moreover, the antileukemia agents all-trans retinoic acid and arsenic trioxide (ATO) were found to reconstitute the silenced B-cell transcriptional program and reduce viability of cHL cell lines. When applied in combination with a screening-identified chemical, ATO evoked reexpression of the CD20 antigen, which could be further therapeutically exploited by enabling CD20 antibody-mediated apoptosis of cHL cells. Furthermore, restoration of the B-cell phenotype also rendered cHL cells susceptible to the B-cell non-Hodgkin lymphoma-tailored small-compound inhibitors ibrutinib and idelalisib. In essence, we report here a conceptually novel, redifferentiation-based treatment strategy for cHL.

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.

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.

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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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