FMP Publications

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

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References
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.

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.

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.

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.

Backbone assignment of perdeuterated proteins by solid-state NMR using proton detection and ultrafast magic-angle spinning
Fricke, P., Chevelkov, V., Zinke, M., Giller(*), K., Becker(*), S.; Lange, A.
Nat Protoc, 12:764-782
(2017)

Tags: Molecular Biophysics (Lange, A.)

Abstract: Solid-state NMR (ssNMR) is a technique that allows the study of protein structure and dynamics at atomic detail. In contrast to X-ray crystallography and cryo-electron microscopy, proteins can be studied under physiological conditions-for example, in a lipid bilayer and at room temperature (0-35 degrees C). However, ssNMR requires considerable amounts (milligram quantities) of isotopically labeled samples. In recent years, 1H-detection of perdeuterated protein samples has been proposed as a method of alleviating the sensitivity issue. Such methods are, however, substantially more demanding to the spectroscopist, as compared with traditional 13C-detected approaches. As a guide, this protocol describes a procedure for the chemical shift assignment of the backbone atoms of proteins in the solid state by 1H-detected ssNMR. It requires a perdeuterated, uniformly 13C- and 15N-labeled protein sample with subsequent proton back-exchange to the labile sites. The sample needs to be spun at a minimum of 40 kHz in the NMR spectrometer. With a minimal set of five 3D NMR spectra, the protein backbone and some of the side-chain atoms can be completely assigned. These spectra correlate resonances within one amino acid residue and between neighboring residues; taken together, these correlations allow for complete chemical shift assignment via a 'backbone walk'. This results in a backbone chemical shift table, which is the basis for further analysis of the protein structure and/or dynamics by ssNMR. Depending on the spectral quality and complexity of the protein, data acquisition and analysis are possible within 2 months.

Complete NMR assignment and conformational analysis of 17-alpha-ethinylestradiol by using RDCs obtained in grafted graphene oxide
Franca(*), J. A., Navarro-Vazquez(*), A., Lei(*), X., Sun, H., Griesinger(*), C.; Hallwass(*), F.
Magn Reson Chem, 55:297-303
(2017)

Tags: Computational Chemistry and Protein Design (Kühne)

Abstract: The 1 H and 13 C NMR spectra of 17-alpha-ethinylestradiol (EE2), a well-known contraceptive, including diastereotopic methylene groups, were fully assigned with the help of residual dipolar couplings (RDC) measured in the recently developed grafted graphene oxide orienting medium. RDC analysis, which included all 1 DCH couplings and the long-range 2 DCH1 H-C identical with13 C coupling, also pointed to the presence of a minor conformation arising from pseudo-rotation of the steroid B ring. Saturation-transfer difference (STD) measurements revealed that the most likely interaction between EE2 and orienting medium occurred on the C and D ring. Copyright (c) 2016 John Wiley & Sons, Ltd.

A Two-Component Adhesive: Tau Fibrils Arise from a Combination of a Well-Defined Motif and Conformationally Flexible Interactions
Xiang(*), S. Q., Kulminskaya(*), N., Habenstein(*), B., Biernat(*), J., Tepper(*), K., Paulat(*), M., Griesinger(*), C., Becker(*), S., Lange, A., Mandelkow(*), E.; Linser(*), R.
J. Am. Chem. Soc., 139:2639-2646
(2017)

Tags: Molecular Biophysics (Lange, A.)

Abstract: Fibrillar aggregates of A beta and Tau in the brain are the major hallmarks of Alzheimer's disease. Most Tau fibers have a twisted appearance, but the twist can be variable and even absent. This ambiguity, which has also been associated with different phenotypes of tauopathies, has led to controversial assumptions about fibril constitution, and it is unclear to-date what the molecular causes of this polymorphism are. To tackle this question, we used solid-state NMR strategies providing assignments of non-seeded three-repeat-domain Tau(3RD) with an inherent heterogeneity. This is in contrast to the general approach to characterize the most homogeneous preparations by construct truncation or intricate seeding protocols. Here, carbon and nitrogen chemical-shift conservation between fibrils revealed invariable secondary-structure properties, however, with inter-monomer interactions variable among samples. Residues with variable amide shifts are localized mostly to N- and C-terminal regions within the rigid beta structure in the repeat region of Tau(3RD). By contrast, the hexapeptide motif in repeat R3, a crucial motif for fibril formation, shows strikingly low variability of all NMR parameters: Starting as a nucleation site for monomer monomer contacts, this six-residue sequence element also turns into a well-defined structural element upon fibril formation. Given the absence of external causes in vitro, the interplay of structurally differently conserved elements in this protein likely reflects an intrinsic property of Tau fibrils.

Post-translational cleavage of Hv1 in human sperm tunes pH- and voltage-dependent gating
Berger(*), T. K., Fusshöller(*), D. M., Goodwin(*), N., Bönigk(*), W., Müller(*), A., Dokani Khesroshahi(*), N., Brenker(*), C., Wachten(*), D., Krause, E., Kaupp(*), U. B.; Strünker(*), T.
J Physiol, 595:1533-1546
(2017)

Tags: Mass Spectrometry (Krause, E.)

Abstract: KEY POINTS: In human sperm, proton flux across the membrane is controlled by the voltage-gated proton channel Hv1. We show that sperm harbour both Hv1 and an N-terminally cleaved isoform termed Hv1Sper. The pH-control of Hv1Sper and Hv1 is distinctively different. Hv1Sper and Hv1 can form heterodimers that combine features of both constituents. Cleavage and heterodimerization of Hv1 might represent an adaptation to the specific requirements of pH control in sperm. ABSTRACT: In human sperm, the voltage-gated proton channel Hv1 controls the flux of protons across the flagellar membrane. Here, we show that sperm harbour Hv1 and a shorter isoform, termed Hv1Sper. Hv1Sper is generated from Hv1 by removal of 68 amino acids from the N-terminus by post-translational proteolytic cleavage. The pH-dependent gating of the channel isoforms is distinctly different. In both Hv1 and Hv1Sper, the conductance-voltage relationship is determined by the pH difference across the membrane (pH). However, simultaneous changes in intracellular and extracellular pH that leave DeltapH constant strongly shift the activation curve of Hv1Sper but not that of Hv1, demonstrating that cleavage of the N-terminus tunes pH sensing in Hv1. Moreover, we show that Hv1 and Hv1Sper assemble as heterodimers that combine features of both constituents. We suggest that cleavage and heterodimerization of Hv1 represents an adaptation to the specific requirements of pH control in sperm.

Functional Significance of the Signal Peptides of Corticotropin-Releasing Factor Receptors
Schülein, R., Gibert, A.; Rutz, C.
Current molecular pharmacology,
(2017)

Tags: Protein Trafficking (Schülein)

Abstract: The corticotropin releasing factor (CRF) receptors belong to the large family of G protein-coupled receptors (GPCRs) and must be transported to the plasma membrane to function properly. The first step of the intracellular transport of GPCRs is their insertion into the membrane of the endoplasmic reticulum (ER). This process is mediated by the translocon complex of the ER membrane and the signal sequences of the receptors. Most GPCRs possess signal sequences which form part of the mature proteins, the so called signal anchor sequences (usually transmembrane domain 1). The CRF receptors possess instead signal sequences at their extreme N tails which were thought to be cleaved off following integration of the receptors into the ER membrane (signal peptides, SPs, also called cleaved signal sequences). Recent work, however, showed that not all subtypes of CRF receptors stick to this rule. Whereas the corticotropin-releasing factor receptor type 1 (CRF1R) and the corticotropin-releasing factor receptor type 2b (CRF2(b)R) possess conventional SPs which are indeed cleaved off following ER insertion, the SP of the cortictropin-releasing factor receptor type 2a (CRF2(a)R) remains uncleaved. It forms a unique N-terminal domain (pseudo signal peptide, PSP) which has surprising functions beyond the ER level. Its presence not only influences expression levels at the plasma membrane but also receptor homodimerisation and, as a consequence, G protein selectivity. In this mini-review, we summarize the progress in understanding the functions of SPs of CRF receptors. Recent data also allow deriving hypotheses for a physiological significance of these sequences.

Gamma oscillations organize top-down signalling to hypothalamus and enable food seeking
Carus-Cadavieco, M., Gorbati, M., Ye(*), L., Bender, F., van der Veldt, S., Kosse(*), C., Borgers(*), C., Lee(*), S. Y., Ramakrishnan(*), C., Hu, Y., Denisova, N., Ramm, F., Volitaki, E., Burdakov(*), D., Deisseroth(*), K., Ponomarenko, A.; Korotkova, T.
Nature, 542:232-236
(2017)

Tags: Behavioral Neurodynamics (Korotkova/Ponomarenko)

Abstract: Both humans and animals seek primary rewards in the environment, even when such rewards do not correspond to current physiological needs. An example of this is a dissociation between food-seeking behaviour and metabolic needs, a notoriously difficult-to-treat symptom of eating disorders. Feeding relies on distinct cell groups in the hypothalamus, the activity of which also changes in anticipation of feeding onset. The hypothalamus receives strong descending inputs from the lateral septum, which is connected, in turn, with cortical networks, but cognitive regulation of feeding-related behaviours is not yet understood. Cortical cognitive processing involves gamma oscillations, which support memory, attention, cognitive flexibility and sensory responses. These functions contribute crucially to feeding behaviour by unknown neural mechanisms. Here we show that coordinated gamma (30-90 Hz) oscillations in the lateral hypothalamus and upstream brain regions organize food-seeking behaviour in mice. Gamma-rhythmic input to the lateral hypothalamus from somatostatin-positive lateral septum cells evokes food approach without affecting food intake. Inhibitory inputs from the lateral septum enable separate signalling by lateral hypothalamus neurons according to their feeding-related activity, making them fire at distinct phases of the gamma oscillation. Upstream, medial prefrontal cortical projections provide gamma-rhythmic inputs to the lateral septum; these inputs are causally associated with improved performance in a food-rewarded learning task. Overall, our work identifies a top-down pathway that uses gamma synchronization to guide the activity of subcortical networks and to regulate feeding behaviour by dynamic reorganization of functional cell groups in the hypothalamus.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
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