FMP Publications

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

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References
Beta blockers prevent correlation of plasma ACE2 activity with echocardiographic parameters in patients with idiopathic dilated cardiomyopathy
Wang(*), Y., Moreira Mda(*), C., Heringer-Walther(*), S., Schultheiss(*), H. P., Siems, W. E., Wessel(*), N.; Walther(*), T.
Journal of cardiovascular pharmacology, 65:8-12
(2015)

Tags: Biochemical Neurobiology (Siems)

Abstract: Plasma angiotensin-converting enzyme (ACE) 2 activity has been demonstrated to be an independent prognostic marker in Chagas' disease, equally potent as B-type natriuretic peptide. This study aimed to investigate the prognostic potency of circulating ACE2 activity in patients with idiopathic dilated cardiomyopathy (DCM). Blood samples were withdrawn from patients with idiopathic DCM and healthy control subjects. The DCM patients were subdivided into 2 groups according to their New York Heart Association classification. The plasma ACE2 activity was measured by a fluorescence method. Plasma ACE2 activity was significantly increased in DCM patients, correlating with clinical severity. It was correlating with echocardiographic parameters in patients with DCM. Furthermore, plasma ACE2 activity had the potency to predict cardiac death and heart transplantation. However, compared with patients with Chagas' disease, the correlation and predictive value of ACE2 activity in patients with DCM was much less pronounced. Beta blocker treatment in patients with DCM was identified to prevent the association between circulating ACE2 activity and echocardiographic parameters. Although ACE2 activity in blood samples of patients with DCM without beta blockers is potent in correlating with the severity of disease and in predicting death and heart transplantation, its correlation and prediction potency are significantly diminished by beta blocker treatment.

Differences in Signal Activation by LH and hCG are Mediated by the LH/CG Receptor's Extracellular Hinge Region
Grzesik, P., Kreuchwig, A., Rutz, C., Furkert, J., Wiesner, B., Schülein, R., Kleinau(*), G., Gromoll(*), J.; Krause, G.
Front Endocrinol (Lausanne), 6:140
(2015)

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

Abstract: The human lutropin (hLH)/choriogonadotropin (hCG) receptor (LHCGR) can be activated by binding two slightly different gonadotropic glycoprotein hormones, choriogonadotropin (CG) - secreted by the placenta, and lutropin (LH) - produced by the pituitary. They induce different signaling profiles at the LHCGR. This cannot be explained by binding to the receptor's leucine-rich-repeat domain (LRRD), as this binding is similar for the two hormones. We therefore speculate that there are previously unknown differences in the hormone/receptor interaction at the extracellular hinge region, which might help to understand functional differences between the two hormones. We have therefore performed a detailed study of the binding and action of LH and CG at the LHCGR hinge region. We focused on a primate-specific additional exon in the hinge region, which is located between LRRD and the serpentine domain. The segment of the hinge region encoded by exon10 was previously reported to be only relevant to hLH signaling, as the exon10-deletion receptor exhibits decreased hLH signaling, but unchanged hCG signaling. We designed an advanced homology model of the hormone/LHCGR complex, followed by experimental characterization of relevant fragments in the hinge region. In addition, we examined predictions of a helical exon10-encoded conformation by block-wise polyalanine (helix supporting) mutations. These helix preserving modifications showed no effect on hormone-induced signaling. However, introduction of a structure-disturbing double-proline mutant LHCGR-Q303P/E305P within the exon10-helix has, in contrast to exon10-deletion, no impact on hLH, but only on hCG signaling. This opposite effect on signaling by hLH and hCG can be explained by distinct sites of hormone interaction in the hinge region. In conclusion, our analysis provides details of the differences between hLH- and hCG-induced signaling that are mainly determined in the L2-beta loop of the hormones and in the hinge region of the receptor.

A modular toolkit to inhibit proline-rich motif-mediated protein-protein interactions
Opitz, R., Müller, M., Reuter, C., Barone, M., Soicke(*), A., Roske(*), Y., Piotukh, K., Huy(*), P., Beerbaum, M., Wiesner, B., Beyermann, M., Schmieder, P., Freund(*), C., Volkmer, R., Oschkinat, H., Schmalz(*), H. G.; Kühne, R.
Proc Natl Acad Sci U S A, 112:5011-5016
(2015)

Tags: Computational Chemistry and Protein Design (Kühne), NMR-Supported Structural Biology (Oschkinat), Peptide Chemistry (Hackenberger/ Volkmer), Solution NMR (Schmieder), Peptide Chemistry (Beyermann), Cellular Imaging (Wiesner)

Abstract: Small-molecule competitors of protein-protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognition motifs. As proof-of-principle, we developed a small, selective, peptidomimetic inhibitor of Ena/VASP EVH1 domain interactions. Highly invasive MDA MB 231 breast-cancer cells treated with this ligand showed displacement of VASP from focal adhesions, as well as from the front of lamellipodia, and strongly reduced cell invasion. General applicability of our strategy is illustrated by the design of an ErbB4-derived ligand containing two ProM-1 fragments, targeting the yes-associated protein 1 (YAP1)-WW domain with a fivefold higher affinity.

Improved intracellular delivery of peptide- and lipid-nanoplexes by natural glycosides
Weng(*), A., Manunta(*), M. D., Thakur(*), M., Gilabert-Oriol(*), R., Tagalakis(), A. D., Eddaoudi(*), A., Munye(*), M. M., Vink(*), C. A., Wiesner, B., Eichhorst, J., Melzig(*), M. F.; Hart(*), S. L.
J Control Release, 206:75-90
(2015)

Tags: Cellular Imaging (Wiesner)

Abstract: Targeted nanocarriers undergo endocytosis upon binding to their membrane receptors and are transported into cellular compartments such as late endosomes and lysosomes. In gene delivery the genetic material has to escape from the cellular compartments into the cytosol. The process of endosomal escape is one of the most critical steps for successful gene delivery. For this reason synthetic lipids with fusogenic properties such as 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) are integrated into the nanocarriers. In this study we show that a natural, plant derived glycoside (SO1861) from Saponaria officinalis L. greatly improves the efficacy of lipid based as well as non-lipid based targeted nanoplexes consisting of a targeted K16 peptide with a nucleic acid binding domain and plasmid-DNA, minicircle-DNA or small interfering RNA (siRNA). By confocal live cell imaging and single cell analyses, we demonstrate that SO1861 augments the escape of the genetic cargo out of the intracellular compartments into the cytosol. Co-localisation experiments with fluorescence labelled dextran and transferrin indicate that SO1861 induces the release of the genetic cargo out of endosomes and lysosomes. However, the transduction efficacy of a lentivirus based gene delivery system was not augmented. In order to design receptor-targeted nanoplexes (LPD) with improved functional properties, SO1861 was integrated into the lipid matrix of the LPD. The SO1861 sensitized LPD (LPDS) were characterized by dynamic light scattering and transmission electron microscopy. Compared to their LPD counterparts the LPDS-nanoplexes showed a greatly improved gene delivery. As shown by differential scanning calorimetry SO1861 can be easily integrated into the lipid bilayer of glycerophospholipid model membranes. This underlines the great potential of SO1861 as a new transfection multiplier for non-viral gene delivery systems.

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
(2015)

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.

Mode of action of claudin peptidomimetics in the transient opening of cellular tight junction barriers
Staat, C., Coisne(*), C., Dabrowski, S., Stamatovic(*), S. M., Andjelkovic(*), A. V., Wolburg(*), H., Engelhardt(*), B.; Blasig, I. E.
Biomaterials, 54:9-20
(2015)

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

Abstract: In epithelial/endothelial barriers, claudins form tight junctions, seal the paracellular cleft, and limit the uptake of solutes and drugs. The peptidomimetic C1C2 from the C-terminal half of claudin-1's first extracellular loop increases drug delivery through epithelial claudin-1 barriers. However, its molecular and structural mode of action remains unknown. In the present study, >100 muM C1C2 caused paracellular opening of various barriers with different claudin compositions, ranging from epithelial to endothelial cells, preferentially modulating claudin-1 and claudin-5. After 6 h incubation, C1C2 reversibly increased the permeability to molecules of different sizes; this was accompanied by redistribution of claudins and occludin from junctions to cytosol. Internalization of C1C2 in epithelial cells depended on claudin-1 expression and clathrin pathway, whereby most C1C2 was retained in recyclosomes >2 h. In freeze-fracture electron microscopy, C1C2 changed claudin-1 tight junction strands to a more parallel arrangement and claudin-5 strands from E-face to P-face association - drastic and novel effects. In conclusion, C1C2 is largely recycled in the presence of a claudin, which explains the delayed onset of barrier and junction loss, the high peptide concentration required and the long-lasting effect. Epithelial/endothelial barriers are specifically modulated via claudin-1/claudin-5, which can be targeted to improve drug delivery.

Defining a conformational consensus motif in cotransin-sensitive signal sequences: a proteomic and site-directed mutagenesis study
Klein, W., Westendorf, C., Schmidt, A., Conill-Cortes, M., Rutz, C., Blohs, M., Beyermann, M., Protze, J., Krause, G., Krause, E.; Schülein, R.
Plos One, 10:e0120886
(2015)

Tags: Protein Trafficking (Schülein), Mass Spectrometry (Krause, E.), Structural Bioinformatics and Protein Design (Krause, G.), Peptide Chemistry (Beyermann)

Abstract: The cyclodepsipeptide cotransin was described to inhibit the biosynthesis of a small subset of proteins by a signal sequence-discriminatory mechanism at the Sec61 protein-conducting channel. However, it was not clear how selective cotransin is, i.e. how many proteins are sensitive. Moreover, a consensus motif in signal sequences mediating cotransin sensitivity has yet not been described. To address these questions, we performed a proteomic study using cotransin-treated human hepatocellular carcinoma cells and the stable isotope labelling by amino acids in cell culture technique in combination with quantitative mass spectrometry. We used a saturating concentration of cotransin (30 micromolar) to identify also less-sensitive proteins and to discriminate the latter from completely resistant proteins. We found that the biosynthesis of almost all secreted proteins was cotransin-sensitive under these conditions. In contrast, biosynthesis of the majority of the integral membrane proteins was cotransin-resistant. Cotransin sensitivity of signal sequences was neither related to their length nor to their hydrophobicity. Instead, in the case of signal anchor sequences, we identified for the first time a conformational consensus motif mediating cotransin sensitivity.

Identification, classification, and signal amplification capabilities of high-turnover gas binding hosts in ultra-sensitive NMR
Kunth, M., Witte, C., Hennig(*), A.; Schröder, L.
Chem Sci, 6:6069-6075
(2015)

Tags: Molecular Imaging (Schröder)

Abstract: Nuclear Magnetic Resonance (NMR) can be a powerful tool for investigating exchange kinetics of host-guest interactions in solution. Beyond conventional direct NMR detection, radiofrequency (RF) saturation transfer can be used to enhance the study of such chemical exchange or to enable signal amplification from a dilute host. However, systems that are both dilute and labile (fast dissociation/re-association) impose specific challenges to direct as well as saturation transfer detection. Here we investigate host-guest systems under previously inaccessible conditions using saturation transfer techniques in combination with hyperpolarized nuclei and quantitative evaluation under different RF exposure. We further use that information to illustrate the consequences for signal amplification capabilities and correct interpretation of observed signal contrast from comparative exchange data of different types of hosts. In particular, we compare binding of xenon (Xe) to cucurbit[6]uril (CB6) with binding to cryptophane-A monoacid (CrA) in water as two different model systems. The Xe complexation with CB6 is extremely difficult to access by conventional NMR due to its low water solubility. We successfully quantified the exchange kinetics of this system and found that the absence of Xe signals related to encapsulated Xe in conventional hyperpolarized Xe-129 NMR is due to line broadening and not due to low binding. By introducing a measure for the gas turnover during constant association-dissociation, we demonstrate that the signal amplification from a dilute pool of CB6 can turn this host into a very powerful contrast agent for Xe MRI applications (100-fold more efficient than cryptophane). However, labile systems only provide improved signal amplification for suitable saturation conditions and otherwise become disadvantageous. The method is applicable to many hosts where Xe is a suitable spy nucleus to probe for non-covalent interactions and should foster reinvestigation of several systems to delineate true absence of interaction from labile complex formation.

Continuous-wave saturation considerations for efficient xenon depolarization
Kunth, M., Witte, C.; Schröder, L.
Nmr Biomed, 28:601-606
(2015)

Tags: Molecular Imaging (Schröder)

Abstract: The combination of hyperpolarized Xe with chemical exchange saturation transfer (Hyper-CEST) is a powerful NMR technique to detect highly dilute concentrations of Xe binding sites using RF saturation pulses. Crucially, that combination of saturation pulse strength and duration that generates the maximal Hyper-CEST effect is a priori unknown. In contrast to CEST in proton MRI, where the system reaches a steady-state for long saturation times, Hyper-CEST has an optimal saturation time, i.e. saturating for shorter or longer reduces the Hyper-CEST effect. Here, we derive expressions for this optimal saturation pulse length. We also found that a pulse strength, B1, corresponding to five times the Xe exchange rate, k(BA) (i.e. B1 = 5 k(BA)/gamma with the gyromagnetic ratio of (129)Xe, gamma), generates directly and without further optimization 96% of the maximal Hyper-CEST contrast while preserving spectral selectivity. As a measure that optimizes the amplitude and the width of the Hyper-CEST response simultaneously, we found an optimal saturation pulse strength corresponding to radical2 times the Xe exchange rate, i.e. B1= radical2k(BA)/gamma. When extremely low host concentration is detected, then the expression for the optimum saturation time simplifies as it approaches the longitudinal relaxation time of free Xe.

Chapter 16 Caged Xenon in Phospholipid Membrane Environments
Schnurr, M., Witte, C.; Schröder, L.
page 288-300.
Publisher: The Royal Society of Chemistry,
(2015)
OA-Link

Tags: Molecular Imaging (Schröder)

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