FMP Publications

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

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In tight junctions, claudins regulate the interactions between occludin, tricellulin and marvelD3, which, inversely, modulate claudin oligomerization
Cording, J., Berg, J., Käding, N., Bellmann, C., Tscheik, C., Westphal(*), J. K., Milatz(*), S., Günzel(*), D., Wolburg(*), H., Piontek, J., Huber(*), O.; Blasig, I. E.
J Cell Sci, 126:554-564

Tags: Molecular and Cell Physiology (Blasig, IE)

Abstract: Tight junctions seal the paracellular cleft of epithelia and endothelia, form vital barriers between tissue compartments and consist of tight-junction-associated marvel proteins (TAMPs) and claudins. The function of TAMPs and the interaction with claudins are not understood. We therefore investigated the binding between the TAMPs occludin, tricellulin, and marvelD3 and their interaction with claudins in living tight-junction-free human embryonic kidney-293 cells. In contrast to claudins and occludin, tricellulin and marvelD3 showed no enrichment at cell-cell contacts indicating lack of homophilic trans-interaction between two opposing cell membranes. However, occludin, marvelD3 and tricellulin exhibited homophilic cis-interactions, along one plasma membrane, as measured by fluorescence resonance energy transfer. MarvelD3 also cis-interacted with occludin and tricellulin heterophilically. Classic claudins, such as claudin-1 to -5 may show cis-oligomerization with TAMPs, whereas the non-classic claudin-11 did not. Claudin-1 and -5 improved enrichment of occludin and tricellulin at cell-cell contacts. The low mobile claudin-1 reduced the membrane mobility of the highly mobile occludin and tricellulin, as studied by fluorescence recovery after photobleaching. Co-transfection of claudin-1 with TAMPs led to changes of the tight junction strand network of this claudin to a more physiological morphology, depicted by freeze-fracture electron microscopy. The results demonstrate multilateral interactions between the tight junction proteins, in which claudins determine the function of TAMPs and vice versa, and provide deeper insights into the tight junction assembly.

Trends in drug delivery through tissue barriers containing tight junctions
Tscheik, C., Blasig, I. E.; Winkler, L.
Tissue Barriers, 1:e24565

Tags: Molecular and Cell Physiology (Blasig, IE)

Abstract: A limitation in the uptake of many drugs is the restricted permeation through tissue barriers. There are two general ways to cross barriers formed by cell layers: by transcytosis or by diffusion through the intercellular space. In the latter, tight junctions (TJs) play the decisive role in the regulation of the barrier permeability. Thus, transient modulation of TJs is a potent strategy to improve drug delivery. There have been extensive studies on surfactant-like absorption enhancers. One of the most effective enhancers found is sodium caprate. However, this modulates TJs in an unspecific fashion. A novel approach would be the specific modulation of TJ-associated marvel proteins and claudins, which are the main structural components of the TJs. Recent studies have identified synthetic peptidomimetics and RNA interference techniques to downregulate the expression of targeted TJ proteins. This review summarizes current progress and discusses the impact on TJs' barrier function.

The mechanism of denaturation and the unfolded state of the alpha-helical membrane-associated protein Mistic
Jacso, T., Bardiaux, B., Broecker(*), J., Fiedler(*), S., Baerwinkel(*), T., Mainz, A., Fink, U., Vargas(*), C., Oschkinat, H., Keller(*), S.; Reif, B.
J Am Chem Soc, 135:18884-18891

Tags: Solid-State NMR Spectroscopy (Reif), NMR-Supported Structure Biology (Oschkinat)

Abstract: In vitro protein-folding studies using chemical denaturants such as urea are indispensible in elucidating the forces and mechanisms determining the stability, structure, and dynamics of water-soluble proteins. By contrast, alpha-helical membrane-associated proteins largely evade such approaches because they are resilient to extensive unfolding. We have used optical and NMR spectroscopy to provide an atomistic-level dissection of the effects of urea on the structure and dynamics of the alpha-helical membrane-associated protein Mistic as well as its interactions with detergent and solvent molecules. In the presence of the zwitterionic detergent lauryl dimethylamine oxide, increasing concentrations of urea result in a complex sequence of conformational changes that go beyond simple two-state unfolding. Exploiting this finding, we report the first high-resolution structural models of the urea denaturation process of an alpha-helical membrane-associated protein and its completely unfolded state, which contains almost no regular secondary structure but nevertheless retains a topology close to that of the folded state.

Efficient alpha-helix induction in a linear peptide chain by N-capping with a bridged-tricyclic diproline analogue
Hack(*), V., Reuter(*), C., Opitz, R., Schmieder, P., Beyermann, M., Neudörfl(*), J. M., Kühne, R.; Schmalz(*), H. G.
Angew Chem Int Ed Engl, 52:9539-9543

Tags: Solution NMR (Schmieder), Peptide Synthesis (Beyermann), Computational Chemistry/Drug Design (Kühne)

Functional properties of cell-free expressed human endothelin A and endothelin B receptors in artificial membrane environments
Proverbio(*), D., Roos(*), C., Beyermann, M., Orban(*), E., Dötsch(*), V.; Bernhard(*), F.
Bba-Biomembranes, 1828:2182-2192

Tags: Peptide Chemistry (Beyermann)

Abstract: The human endothelin receptors are members of the rhodopsin class A of G-protein coupled receptors and key modulators of blood pressure regulation. Their functional in vitro characterization has widely been limited by the availability of high quality samples. We have optimized cell-free expression protocols for the human endothelin A and endothelin B receptors by implementing co-translational association approaches of the synthesized proteins with supplied liposomes or nanodiscs. Efficiency of membrane association and ligand binding properties of the receptors have systematically been studied in correlation to different membrane environments and lipid types. Ligand binding was analyzed by a number of complementary assays including radioassays, surface plasmon resonance and fluorescence measurements. High affinity binding of the peptide ligand ET-I to both endothelin receptors could be obtained with several conditions and the highest Bmax values were measured In association with nanodiscs. We could further obtain the characteristic differential binding pattern of the two endothelin receptors with a panel of selected agonists and antagonists. Two intrinsic properties of the functionally folded endothelin B receptor, the proteolytic processing based on conformational recognition as well as the formation of SDS-resistant complexes with the peptide ligand ET-1, were observed with samples obtained from several cell-free expression conditions. High affinity and specific binding of ligands could furthermore be obtained with non-purified receptor samples in crude cell-free reaction mixtures, thus providing new perspectives for fast in vitro screening applications. (C) 2013 Elsevier B.V. All rights reserved.

Structural insights into the mechanism of GTPase activation in the GIMAP family
Schwefel(*), D., Arasu(*), B. S., Marino(*), S. F., Lamprecht(*), B., Kochert(*), K., Rosenbaum(*), E., Eichhorst, J., Wiesner, B., Behlke(*), J., Rocks(*), O., Mathas(*), S.; Daumke(*), O.
Structure (London, England : 1993), 21:550-559

Tags: Cellular Imaging (Wiesner)

Abstract: GTPases of immunity-associated proteins (GIMAPs) are regulators of lymphocyte survival and homeostasis. We previously determined the structural basis of GTP-dependent GIMAP2 scaffold formation on lipid droplets. To understand how its GTP hydrolysis is activated, we screened for other GIMAPs on lipid droplets and identified GIMAP7. In contrast to GIMAP2, GIMAP7 displayed dimerization-stimulated GTP hydrolysis. The crystal structure of GTP-bound GIMAP7 showed a homodimer that assembled via the G domains, with the helical extensions protruding in opposite directions. We identified a catalytic arginine that is supplied to the opposing monomer to stimulate GTP hydrolysis. GIMAP7 also stimulated GTP hydrolysis by GIMAP2 via an analogous mechanism. Finally, we found GIMAP2 and GIMAP7 expression differentially regulated in several human T cell lymphoma lines. Our findings suggest that GTPase activity in the GIMAP family is controlled by homo- and heterodimerization. This may have implications for the differential roles of some GIMAPs in lymphocyte survival.

Stabilization of peptides for intracellular applications by phosphoramidate-linked polyethylene glycol chains
Nischan, N., Chakrabarti(*), A., Serwa, R. A., Bovee-Geurts(*), P. H., Brock(*), R.; Hackenberger, C. P.
Angew Chem Int Ed Engl, 52:11920-11924

Tags: Chemical Biology II (Hackenberger)

Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts
Theillet, F. X., Rose, H. M., Liokatis, S., Binolfi, A., Thongwichian, R., Stuiver, M.; Selenko, P.
Nat Protoc, 8:1416-1432

Tags: In-Cell NMR (Selenko)

Abstract: We outline NMR protocols for site-specific mapping and time-resolved monitoring of protein phosphorylation reactions using purified kinases and mammalian cell extracts. These approaches are particularly amenable to intrinsically disordered proteins and unfolded, regulatory protein domains. We present examples for the (1)(5)N isotope-labeled N-terminal transactivation domain of human p53, which is either sequentially reacted with recombinant enzymes or directly added to mammalian cell extracts and phosphorylated by endogenous kinases. Phosphorylation reactions with purified enzymes are set up in minutes, whereas NMR samples in cell extracts are prepared within 1 h. Time-resolved NMR measurements are performed over minutes to hours depending on the activities of the probed kinases. Phosphorylation is quantitatively monitored with consecutive 2D (1)H-(1)(5)N band-selective optimized-flip-angle short-transient (SOFAST)-heteronuclear multiple-quantum (HMQC) NMR experiments, which provide atomic-resolution insights into the phosphorylation levels of individual substrate residues and time-dependent changes thereof, thereby offering unique advantages over western blotting and mass spectrometry.

A Supramolecular Peptide Synthesizer
Bertran-Vicente, J.; Hackenberger, C. P. R.
Angew Chem Int Edit, 52:6140-6142

Tags: Chemical Biology II (Hackenberger)

KSHV ORF67 encoded lytic protein localizes on the nuclear membrane and alters emerin distribution
Farina(*), A., Santarelli(*), R., Bloise(*), R., Gonnella(*), R., Granato(*), M., Bei(*), R., Modesti(*), A., Cirone(*), M., Bengtsson, L., Angeloni(*), A.; Faggioni(*), A.
Virus Res, 175:143-150

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: p29, a newly identified Kaposi's sarcoma-associated herpesvirus (KSHV) protein, is the product of ORF67, the positional homolog of the conserved herpesvirus protein UL34. Like its homologues in other herpesviruses, p29 is expressed early during viral lytic cycle, and is localized on the nuclear rim. Upon chemical induction of viral replication in primary effusion lymphoma cells, p29 interacts with p33, encoded by ORF69, the positional homolog of the conserved herpesvirus protein UL31, and both proteins colocalize on the nuclear membrane. IFA and biochemical analysis of infected or transfected cells showed that p29 expression resulted in delocalization and hyperphosphorylation of emerin, whereas other nuclear lamin associated proteins, such as LUMA, LB1 and LBR were not affected. Mislocalization of emerin was robustly increased upon combined expression of p29 and p33, suggesting that emerin destabilization might represent the first step in nuclear lamina disassembling, a process necessary for nucleocapsid maturation. (C) 2013 Elsevier B.V. All rights reserved.

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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 
+4930 94793 - 109 (Fax)

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