FMP Publications

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

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Redox Regulation of Cell Contacts by Tricellulin and Occludin: Redox-Sensitive Cysteine Sites in Tricellulin Regulate Both Tri- and Bicellular Junctions in Tissue Barriers as Shown in Hypoxia and Ischemia
Cording, J., Günther, R., Vigolo(*), E., Tscheik, C., Winkler, L., Schlattner, I., Lorenz, D., Haseloff, R. F., Schmidt-Ott(*), K. M., Wolburg(*), H.; Blasig, I. E.
Antioxid Redox Signal, 23:1035-1049

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

Abstract: UNLABELLED: Tight junctions (TJs) seal paracellular clefts in epithelia/endothelia and form tissue barriers for proper organ function. TJ-associated marvel proteins (TAMPs; tricellulin, occludin, marvelD3) are thought to be relevant to regulation. Under normal conditions, tricellulin tightens tricellular junctions against macromolecules. Traces of tricellulin occur in bicellular junctions. AIMS: As pathological disturbances have not been analyzed, the structure and function of human tricellulin, including potentially redox-sensitive Cys sites, were investigated under reducing/oxidizing conditions at 3- and 2-cell contacts. RESULTS: Ischemia, hypoxia, and reductants redistributed tricellulin from 3- to 2-cell contacts. The extracellular loop 2 (ECL2; conserved Cys321, Cys335) trans-oligomerized between three opposing cells. Substitutions of these residues caused bicellular localization. Cys362 in transmembrane domain 4 contributed to bicellular heterophilic cis-interactions along the cell membrane with claudin-1 and marvelD3, while Cys395 in the cytosolic C-terminal tail promoted homophilic tricellullar cis-interactions. The Cys sites included in homo-/heterophilic bi-/tricellular cis-/trans-interactions contributed to cell barrier tightness for small/large molecules. INNOVATION: Tricellulin forms TJs via trans- and cis-association in 3-cell contacts, as demonstrated electron and quantified fluorescence microscopically; it tightens 3- and 2-cell contacts. Tricellulin's ECL2 specifically seals 3-cell contacts redox dependently; a structural model is proposed. CONCLUSIONS: TAMP ECL2 and claudins' ECL1 share functionally and structurally similar features involved in homo-/heterophilic tightening of cell-cell contacts. Tricellulin is a specific redox sensor and sealing element at 3-cell contacts and may compensate as a redox mediator for occludin loss at 2-cell contacts in vivo and in vitro. Molecular interaction mechanisms were proposed that contribute to tricellulin's function. In conclusion, tricellulin is a junctional redox regulator for ischemia-related alterations.

Redox-sensitive structure and function of the first extracellular loop of the cell-cell contact protein claudin-1: lessons from molecular structure to animals
Dabrowski, S., Staat, C., Zwanziger, D., Sauer(*), R. S., Bellmann, C., Günther, R., Krause, E., Haseloff, R. F., Rittner(*), H.; Blasig, I. E.
Antioxid Redox Signal, 22:1-14

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

Abstract: UNLABELLED: The paracellular cleft within epithelia/endothelia is sealed by tight junction (TJ) proteins. Their extracellular loops (ECLs) are assumed to control paracellular permeability and are targets of pathogenes. We demonstrated that claudin-1 is crucial for paracellular tightening. Its ECL1 is essential for the sealing and contains two cysteines conserved throughout all claudins. AIMS: We prove the hypothesis that this cysteine motif forms a redox-sensitive intramolecular disulfide bridge and, hence, the claudin-1-ECL1 constitutes a functional structure which is associated to ECLs of this and other TJ proteins. RESULTS: The structure and function of claudin-1-ECL1 was elucidated by investigating sequences of this ECL as synthetic peptides, C1C2, and as recombinant proteins, and exhibited a beta-sheet binding surface flanked by an alpha-helix. These sequences bound to different claudins, their ECL1, and peptides with nanomolar binding constants. C-terminally truncated C1C2 (-4aaC) opened cellular barriers and the perineurium. Recombinant ECL1 formed oligomers, and bound to claudin-1 expressing cells. Oligomerization and claudin association were abolished by reducing agents, indicating intraloop disulfide bridging and redox sensitivity. INNOVATION: The structural and functional model based on our in vitro and in vivo investigations suggested that claudin-1-ECL1 constitutes a functional and ECL-binding beta-sheet, stabilized by a shielded and redox-sensitive disulfide bond. CONCLUSION: Since the beta-sheet represents a consensus sequence of claudins and further junctional proteins, a general structural feature is implied. Therefore, our model is of general relevance for the TJ assembly in normal and pathological conditions. C1C2-4aaC is a new drug enhancer that is used to improve pharmacological treatment through tissue barriers.

Phenothiazine-derived antipsychotic drugs inhibit dynamin and clathrin-mediated endocytosis
Daniel(*), J. A., Chau(*), N., Abdel-Hamid(*), M. K., Hu(*), L., von Kleist, L., Whiting(*), A., Krishnan(*), S., Maamary(*), P., Joseph(*), S. R., Simpson(*), F., Haucke, V., McCluskey(*), A.; Robinson(*), P. J.
Traffic, 16:635-654

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Chlorpromazine is a phenothiazine-derived antipsychotic drug (APD) that inhibits clathrin-mediated endocytosis (CME) in cells by an unknown mechanism. We examined whether its action and that of other APDs might be mediated by the GTPase activity of dynamin. Eight of eight phenothiazine-derived APDs inhibited dynamin I (dynI) in the 2-12 microm range, the most potent being trifluoperazine (IC50 2.6 +/- 0.7 microm). They also inhibited dynamin II (dynII) at similar concentrations. Typical and atypical APDs not based on the phenothiazine scaffold were 8- to 10-fold less potent (haloperidol and clozapine) or were inactive (droperidol, olanzapine and risperidone). Kinetic analysis showed that phenothiazine-derived APDs were lipid competitive, while haloperidol was uncompetitive with lipid. Accordingly, phenothiazine-derived APDs inhibited dynI GTPase activity stimulated by lipids but not by various SH3 domains. All dynamin-active APDs also inhibited transferrin (Tfn) CME in cells at related potencies. Structure-activity relationships (SAR) revealed dynamin inhibition to be conferred by a substituent group containing a terminal tertiary amino group at the N2 position. Chlorpromazine was previously proposed to target AP-2 recruitment in the formation of clathrin-coated vesicles (CCV). However, neither chlorpromazine nor thioridazine affected AP-2 interaction with amphiphysin or clathrin. Super-resolution microscopy revealed that chlorpromazine blocks neither clathrin recruitment by AP-2, nor AP-2 recruitment, showing that CME inhibition occurs downstream of CCV formation. Overall, potent dynamin inhibition is a shared characteristic of phenothiazine-derived APDs, but not other typical or atypical APDs, and the data indicate that dynamin is their likely in-cell target in endocytosis.

Thermodynamics of protein destabilization in live cells
Danielsson(*), J., Mu(*), X., Lang(*), L., Wang(*), H., Binolfi, A., Theillet, F. X., Bekei, B., Logan(*), D. T., Selenko, P., Wennerstrom(*), H.; Oliveberg(*), M.
Proc Natl Acad Sci U S A, 112:12402-12407

Tags: In-Cell NMR (Selenko)

Abstract: Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a beta-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 degrees C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.

SEPT9 negatively regulates ubiquitin-dependent downregulation of EGFR
Diesenberg, K., Beerbaum, M., Fink, U., Schmieder, P.; Krauss, M.
J Cell Sci, 128:397-407

Tags: Molecular Pharmacology and Cell Biology (Haucke), Solution NMR (Schmieder)

Abstract: Septins constitute a family of GTP-binding proteins that are involved in a variety of biological processes. Several isoforms have been implicated in disease, but the molecular mechanisms underlying pathogenesis are poorly understood. Here, we show that depletion of SEPT9 decreases surface levels of epidermal growth factor receptors (EGFRs) by enhancing receptor degradation. We identify a consensus motif within the SEPT9 N-terminal domain that supports its association with the adaptor protein CIN85 (also known as SH3KBP1). We further show CIN85-SEPT9 to be localized exclusively to the plasma membrane, where SEPT9 is recruited to EGF-engaged receptors in a CIN85-dependent manner. Finally, we demonstrate that SEPT9 negatively regulates EGFR degradation by preventing the association of the ubiquitin ligase Cbl with CIN85, resulting in reduced EGFR ubiquitylation. Taken together, these data provide a mechanistic explanation of how SEPT9, though acting exclusively at the plasma membrane, impairs the sorting of EGFRs into the degradative pathway.

Probing heterobivalent binding to the endocytic AP-2 adaptor complex by DNA-based spatial screening
Diezmann(*), F., von Kleist, L., Haucke, V.; Seitz(*), O.
Organic & Biomolecular Chemistry, 13:8008-8015

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: The double helical DNA scaffold offers a unique set of properties, which are particularly useful for studies of multivalency in biomolecular interactions: (i) multivalent ligand displays can be formed upon nucleic acid hybridization in a self-assembly process, which facilitates spatial screening (ii) valency and spatial arrangement of the ligand display can be precisely controlled and (iii) the flexibility of the ligand display can be adjusted by integrating nick sites and unpaired template regions. Herein we describe the use of DNA-based spatial screening for the characterization of the adaptor complex 2 (AP-2), a central interaction hub within the endocytic protein network in clathrin-mediated endocytosis. AP-2 is comprised of a core domain and two, so-called appendage domains, the alpha- and the beta 2-ear, which associate with cyto-plasmatic proteins required for the formation or maturation of clathrin/AP-2 coated pits. Each appendage domain has two binding grooves which recognize distinct peptide motives with micromolar affinity. This provides opportunities for enhanced interactions with protein molecules that contain two (or more) different peptide motives. To determine whether a particular, spatial arrangement of binding motifs is required for high affinity binding we probed the distance-affinity relationships by means of DNA-programmed spatial screening with self-assembled peptide-DNA complexes. By using trimolecular and tetra-molecular assemblies two different peptides were positioned in 2-22 nucleotide distance. The binding data obtained with both recombinant protein in well-defined buffer systems and native AP-2 in brain extract suggests that the two binding sites of the AP-2 alpha-appendage can cooperate to provide up to 40-fold enhancement of affinity compared to the monovalent interaction. The distance between the two recognized peptide motives was less important provided that the DNA duplex segments were connected by flexible, single strand segments. By contrast, the experiments with a more rigid, duplex-spaced assembly revealed marked distance dependencies. Consequences for the function of adaptor proteins are discussed.

Lipids, GTPases, and their regulators in membrane dynamics: an intracellular menage a trois
Echard(*), A.; Haucke, V.
Mol Biol Cell, 26:1012-1013

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Postsynaptic D2 dopamine receptor supersensitivity in the striatum of mice lacking TAAR1
Espinoza(*), S., Ghisi, V., Emanuele(*), M., Leo(*), D., Sukhanov(*), I., Sotnikova(*), T. D., Chieregatti(*), E.; Gainetdinov(*), R. R.
Neuropharmacology, 93:308-313

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: Trace Amine-Associated Receptor 1 (TAAR1) is a G protein-coupled receptor (GPCR) known to modulate dopaminergic system through several mechanisms. Mice lacking this receptor show a higher sensitivity to dopaminergic stimuli, such as amphetamine; however, it is not clear whether D1 or D2 dopamine receptors and which associated intracellular signaling events are involved in this modulation. In the striatum of TAAR1 knock out (TAAR1-KO mice) we found that D2, but not D1, dopamine receptors were over-expressed, both in terms of mRNA and protein levels. Moreover, the D2 dopamine receptor-related G protein-independent AKT/GSK3 signaling pathway was selectively activated, as indicated by the decrease of phosphorylation of AKT and GSK3beta. The decrease in phospho-AKT levels, suggesting an increase in D2 dopamine receptor activity in basal conditions, was associated with an increase of AKT/PP2A complex, as revealed by co-immunoprecipitation experiments. Finally, we found that the locomotor activation induced by the D2 dopamine receptor agonist quinpirole, but not by the full D1 dopamine receptor agonist SKF-82958, was increased in TAAR1-KO mice. These data demonstrate pronounced supersensitivity of postsynaptic D2 dopamine receptors in the striatum of TAAR1-KO mice and indicate that a close interaction of TAAR1 and D2 dopamine receptors at the level of postsynaptic structures has important functional consequences.

Specific 13C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies
Fasshuber, H. K., Demers, J. P., Chevelkov, V., Giller(*), K., Becker(*), S.; Lange, A.
J Magn Reson, 252:10-19

Tags: Molecular Biophysics (Lange, A.)

Abstract: Here we present an isotopic labeling strategy to easily obtain unambiguous long-range distance restraints in protein solid-state NMR studies. The method is based on the inclusion of two biosynthetic precursors in the bacterial growth medium, alpha-ketoisovalerate and alpha-ketobutyrate, leading to the production of leucine, valine and isoleucine residues that are exclusively (13)C labeled on methyl groups. The resulting spectral simplification facilitates the collection of distance restraints, the verification of carbon chemical shift assignments and the measurement of methyl group dynamics. This approach is demonstrated on the type-three secretion system needle of Shigella flexneri, where 49 methyl-methyl and methyl-nitrogen distance restraints including 10 unambiguous long-range distance restraints could be collected. By combining this labeling scheme with ultra-fast MAS and proton detection, the assignment of methyl proton chemical shifts was achieved.

Structural heterogeneity in microcrystalline ubiquitin studied by solid-state NMR
Fasshuber, H. K., Lakomek(*), N. A., Habenstein(*), B., Loquet(*), A., Shi, C., Giller(*), K., Wolff(*), S., Becker(*), S.; Lange, A.
Protein Sci, 24:592-598

Tags: Molecular Biophysics (Lange, A.)

Abstract: By applying [1-(13) C]- and [2-(13) C]-glucose labeling schemes to the folded globular protein ubiquitin, a strong reduction of spectral crowding and increase in resolution in solid-state NMR (ssNMR) spectra could be achieved. This allowed spectral resonance assignment in a straightforward manner and the collection of a wealth of long-range distance information. A high precision solid-state NMR structure of microcrystalline ubiquitin was calculated with a backbone rmsd of 1.57 to the X-ray structure and 1.32 A to the solution NMR structure. Interestingly, we can resolve structural heterogeneity as the presence of three slightly different conformations. Structural heterogeneity is most significant for the loop region beta1-beta2 but also for beta-strands beta1, beta2, beta3, and beta5 as well as for the loop connecting alpha1 and beta3. This structural polymorphism observed in the solid-state NMR spectra coincides with regions that showed dynamics in solution NMR experiments on different timescales.

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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 
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