FMP Publications

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

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References
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.

Claudins are essential for cell shape changes and convergent extension movements during neural tube closure
Baumholtz(*), A. I., Simard(*), A., Nikolopoulou(*), E., Oosenbrug(*), M., Collins(*), M. M., Piontek, A., Krause, G., Piontek(*), J., Greene(*), N. D. E.; Ryan(*), A. K.
Developmental biology, 428:25-38
(2017)

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

Abstract: During neural tube closure, regulated changes at the level of individual cells are translated into large-scale morphogenetic movements to facilitate conversion of the flat neural plate into a closed tube. Throughout this process, the integrity of the neural epithelium is maintained via cell interactions through intercellular junctions, including apical tight junctions. Members of the claudin family of tight junction proteins regulate paracellular permeability, apical-basal cell polarity and link the tight junction to the actin cytoskeleton. Here, we show that claudins are essential for neural tube closure: the simultaneous removal of Cldn3, -4 and -8 from tight junctions caused folate-resistant open neural tube defects. Their removal did not affect cell type differentiation, neural ectoderm patterning nor overall apical-basal polarity. However, apical accumulation of Vangl2, RhoA, and pMLC were reduced, and Par3 and Cdc42 were mislocalized at the apical cell surface. Our data showed that claudins act upstream of planar cell polarity and RhoA/ROCK signaling to regulate cell intercalation and actin-myosin contraction, which are required for convergent extension and apical constriction during neural tube closure, respectively.

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.

Structural determinants of a conserved enantiomer-selective carvone binding pocket in the human odorant receptor OR1A1
Geithe(*), C., Protze, J., Kreuchwig, F., Krause, G.; Krautwurst(*), D.
Cellular and molecular life sciences : CMLS,
(2017)

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

Abstract: Chirality is a common phenomenon within odorants. Most pairs of enantiomers show only moderate differences in odor quality. One example for enantiomers that are easily discriminated by their odor quality is the carvones: humans significantly distinguish between the spearmint-like (R)-(-)-carvone and caraway-like (S)-(+)-carvone enantiomers. Moreover, for the (R)-(-)-carvone, an anosmia is observed in about 8% of the population, suggesting enantioselective odorant receptors (ORs). With only about 15% de-orphaned human ORs, the lack of OR crystal structures, and few comprehensive studies combining in silico and experimental approaches to elucidate structure-function relations of ORs, knowledge on cognate odorant/OR interactions is still sparse. An adjusted homology modeling approach considering OR-specific proline-caused conformations, odorant docking studies, single-nucleotide polymorphism (SNP) analysis, site-directed mutagenesis, and subsequent functional studies with recombinant ORs in a cell-based, real-time luminescence assay revealed 11 amino acid positions to constitute an enantioselective binding pocket necessary for a carvone function in human OR1A1 and murine Olfr43, respectively. Here, we identified enantioselective molecular determinants in both ORs that discriminate between minty and caraway odor. Comparison with orthologs from 36 mammalian species demonstrated a hominid-specific carvone binding pocket with about 100% conservation. Moreover, we identified loss-of-function SNPs associated with the carvone binding pocket of OR1A1. Given carvone enantiomer-specific receptor activation patterns including OR1A1, our data suggest OR1A1 as a candidate receptor for constituting a carvone enantioselective phenotype, which may help to explain mechanisms underlying a (R)-(-)-carvone-specific anosmia in humans.

Molecular features of the L-type amino acid transporter 2 determine different import and export profiles for thyroid hormones and amino acids
Hinz, K. M., Neef, D., Rutz, C., Furkert, J., Köhrle(*), J., Schülein, R.; Krause, G.
Mol Cell Endocrinol, 443:163-174
(2017)

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

Abstract: The L-type amino acid transporter 2 (LAT2) imports amino acids (AA) and also certain thyroid hormones (TH), e.g. 3,3'-T2 and T3, but not rT3 and T4. We utilized LAT2 mutations (Y130A, N133S, F242W) that increase 3,3'-T2 import and focus here on import and export capacity for AA, T4, T3, BCH and derivatives thereof to delineate molecular features. Transport studies and analysis of competitive inhibition of import by radiolabelled TH and AA were performed in Xenopus laevis oocytes. Only Y130A, a pocket widening mutation, enabled import for T4 and increased it for T3. Mutant F242W showed increased 3,3'-T2 import but no import rates for other TH derivatives. No export was detected for any TH by LAT2-wild type (WT). Mutations Y130A and N133S enabled only the export of 3,3'-T2, while N133S also increased AA export. Thus, distinct molecular LAT2-features determine bidirectional AA transport but only an unidirectional 3,3'-T2 and T3 import.

Structural-Functional Features of the Thyrotropin Receptor: A Class A G-Protein-Coupled Receptor at Work
Kleinau(*), G., Worth, C. L., Kreuchwig, A., Biebermann(*), H., Marcinkowski, P., Scheerer(*), P.; Krause, G.
Front Endocrinol (Lausanne), 8:86
(2017)

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

Abstract: The thyroid-stimulating hormone receptor (TSHR) is a member of the glycoprotein hormone receptors, a sub-group of class A G-protein-coupled receptors (GPCRs). TSHR and its endogenous ligand thyrotropin (TSH) are of essential importance for growth and function of the thyroid gland and proper function of the TSH/TSHR system is pivotal for production and release of thyroid hormones. This receptor is also important with respect to pathophysiology, such as autoimmune (including ophthalmopathy) or non-autoimmune thyroid dysfunctions and cancer development. Pharmacological interventions directly targeting the TSHR should provide benefits to disease treatment compared to currently available therapies of dysfunctions associated with the TSHR or the thyroid gland. Upon TSHR activation, the molecular events conveying conformational changes from the extra- to the intracellular side of the cell across the membrane comprise reception, conversion, and amplification of the signal. These steps are highly dependent on structural features of this receptor and its intermolecular interaction partners, e.g., TSH, antibodies, small molecules, G-proteins, or arrestin. For better understanding of signal transduction, pathogenic mechanisms such as autoantibody action and mutational modifications or for developing new pharmacological strategies, it is essential to combine available structural data with functional information to generate homology models of the entire receptor. Although so far these insights are fragmental, in the past few decades essential contributions have been made to investigate in-depth the involved determinants, such as by structure determination via X-ray crystallography. This review summarizes available knowledge (as of December 2016) concerning the TSHR protein structure, associated functional aspects, and based on these insights we suggest several receptor complex models. Moreover, distinct TSHR properties will be highlighted in comparison to other class A GPCRs to understand the molecular activation mechanisms of this receptor comprehensively. Finally, limitations of current knowledge and lack of information are discussed highlighting the need for intensified efforts toward TSHR structure elucidation.

Thyroid hormone transport across L-type amino acid transporters: What can molecular modelling tell us?
Krause, G.; Hinz, K. M.
Mol Cell Endocrinol,
(2017)

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

Abstract: Thyroid hormones (THs) and their derivatives require transmembrane transporters (TTs) to mediate their translocation across the cell membrane. Among these TTs, the L-type amino acid transporters (LAT) not only transport amino acids (AAs) but also certain THs and their derivatives. This review summarizes available knowledge concerning structure function patterns of the TH transport by LAT1 and LAT2. For example, LAT2 imports 3,3'-T2 and T3, but not rT3 and T4. In contrast to amino acids, THs are not at all exported by LAT2. Homology modelling of LAT1 and LAT2 is based on available crystal structures from the same superfamily the amino acid/polyamine/organocation transporter (APC). Molecular model guided mutagenesis has been used to predict substrate interaction sites. A common recognition feature for amino acid- and TH-derivatives has been suggested in an interior cavity of LAT1 and LAT2. Therein additional distinct molecular determinants that are responsible for the bidirectional AA transport but allowing only unidirectional import of particular THs have been confirmed for LAT2 by mutagenesis. Characterized substrate features that are needed for TH translocation and distinct LAT2 properties will be highlighted to understand the molecular import and export mechanisms of this transporter in more detail.

Polar and charged extracellular residues conserved among barrier-forming claudins contribute to tight junction strand formation
Piontek, A., Rossa, J., Protze, J., Wolburg(*), H., Hempel(*), C., Günzel(*), D., Krause, G.; Piontek(*), J.
Annals of the New York Academy of Sciences,
(2017)

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

Abstract: Claudins (Cldn) form the backbone of tight junction (TJ) strands and thereby regulate paracellular permeability for solutes and water. Polymeric strands are formed by homo- and heterophilic cis- and trans-interactions between claudin protomers. Crystal structures of some claudins have been resolved; however, the mechanism by which claudins assemble into TJ strands remains unclear. To elucidate strand architecture, TJ-like strands were reconstituted in HEK293 cells by claudin transfection. Determinants of prototypic, classic barrier-forming claudins (Cldn1, -3, and -5) involved in strand formation were analyzed by mutagenesis. The capability of claudin constructs to interact in trans and to form strands was investigated by cell contact-enrichment assays and freeze-fracture electron microscopy. Residues in extracellular loops 1 and 2 of the claudins affecting strand formation were identified. Using homology modeling and molecular docking, we tested working concepts for the arrangement of claudin protomers within TJ strands. We show that the charge of Lys65 in Cldn1 and Glu158 in Cldn3, but not of Arg30 or Asp145 in Cldn3, and the polarity of Gln56 and Gln62 in Cldn3 and of Gln57 in Cldn5 are necessary for TJ strand formation. These residues are all conserved among barrier-forming classic claudins. The results contribute to mechanistic understanding of claudin-based regulation of paracellular permeability.

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.

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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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