FMP Publications

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

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Triple resonance cross-polarization for more sensitive 13C MAS NMR spectroscopy of deuterated proteins
Akbey, Ü., Camponeschi, F., van Rossum, B. J.; Oschkinat, H.
Chemphyschem, 12:2092-2096

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Save the last WALTZ for me: the use of simultaneous proton and deuterium cross-polarization for (13)C CPMAS NMR spectroscopy in highly deuterated proteins is discussed. The aim of the new method introduced herein, triple-resonance cross-polarization, is to increase the sensitivity of the carbon-detected methods in such systems.

The binding of an amphipathic peptide to lipid monolayers at the air/water interface is modulated by the lipid headgroup structure
Arouri(*), A., Kerth(*), A., Dathe, M.; Blume(*), A.
Langmuir, 27:2811-2818

Tags: Peptide-Lipid-Interaction/ Peptide Transport (Dathe)

Abstract: We used monolayer techniques combined with infrared reflection absorption spectroscopy (IRRAS) to study the behavior of the 18-mer cationic peptide KLA1 (KLAL KLAL KAW KAAL KLA-NH2) at the air/water interface as well as its interaction with lipid films of different composition. The adsorption of the peptide from the subphase to the air/water interface was observed measuring the increase in surface pressure (pi) at constant surface area. The binding of the peptide to lipid monolayers was followed by recording the change in lipid area at a constant surface pressure (pi = 30 mN m(-1)). At the air/water interface, the peptide initially adopted an alpha-helix at large surface area per molecule, that is, low surface pressure, but further accumulation of the peptide at the interface induced a conformational change from alpha-helix to intermolecular beta-sheet, driven by intermolecular aggregation. When the peptide was injected into the subphase underneath lipid monolayers, it adsorbed pronouncedly to anionic monolayers containing phosphatidylglycerol forming an alpha-helix, but not to zwitterionic lipid monolayers. The large change in area observed upon peptide binding suggests that the peptide helix was incorporated into the apolar chain region of the lipids. An apparent partition coefficient of (0.3-1) x 10(6) M(-1) could be calculated for binding to pure POPG monolayers. Significant differences in binding affinity were observed comparing PG/PC with PG/PE monolayers, with the latter showing a higher binding constant. This shows that not only electrostatic and hydrophobic effects but also specific interactions between the headgroups of the lipids and the peptide side chains modulate the binding affinity.

Morphological changes induced by the action of antimicrobial peptides on supported lipid bilayers
Arouri(*), A., Kiessling(*), V., Tamm(*), L., Dathe, M.; Blume(*), A.
J Phys Chem B, 115:158-167

Tags: Peptide-Lipid-Interaction/ Peptide Transport (Dathe)

Abstract: We utilized epifluorescence microscopy to investigate the morphological changes in labeled lipid bilayers supported on quartz surfaces (SLBs) induced by the interaction of cationic antimicrobial peptides with the lipid membranes. The SLBs were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and mixtures thereof as well as from Escherichia coli lipid extract. We succeeded in the preparation of POPG and POPG-rich SLBs without the necessity to use fusogenic agents such as calcium by using the Langmuir-Blodgett/Langmuir-Schaefer transfer method. The adsorption of the peptides to the SLBs was initially driven by electrostatic interactions with the PG headgroups and led to the formation of lipid protrusions bulging out from the lipid layer facing the bulk, originating particularly from domain boundaries and membrane defects. The shape, size, and frequency of the lipid protrusions are mainly controlled by the peptide macroscopic properties and the membrane composition. A restructuring of the lipid protrusions into other structures can also occur over time.

Interaction of W-substituted analogs of cyclo-RRRWFW with bacterial lipopolysaccharides: the role of the aromatic cluster in antimicrobial activity
Bagheri, M., Keller, S.; Dathe, M.
Antimicrob Agents Chemother, 55:788-797

Tags: Peptide -Lipid-Interaction/ Peptide Transport (Dathe)

Abstract: The activity of cyclo-RRRWFW (c-WFW) against Escherichia coli has been shown to be modulated by the aromatic motif and the lipopolysaccharides (LPS) in the bacterial outer membrane. To identify interaction sites and to elucidate the mode of c-WFW action, peptides were synthesized by the replacement of tryptophan (W) with analogs having altered hydrophobicity, dipole and quadrupole moments, hydrogen-bonding ability, amphipathicity, and ring size. The peptide activity against Bacillus subtilis and erythrocytes increased with increasing hydrophobicity, whereas the effect on E. coli revealed a more complex pattern. Although they had no effect on the E. coli inner membrane even at concentrations higher than the MIC, peptides permeabilized the outer membrane according to their antimicrobial activity pattern, suggesting a major role of LPS in peptide transport across the wall. For isothermal titration calorimetry (ITC) studies of peptide-lipid bilayer interaction, we used POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline), either alone or in mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), to mimic the charge properties of eukaryotic and bacterial membranes, respectively, as well as in mixtures with lipid A, rough LPS, and smooth LPS as models of the outer membrane of E. coli. Peptide accumulation was determined by both electrostatic and hydrophobic interactions. The susceptibility of the lipid systems followed the order of POPC-smooth LPS >> POPC-rough LPS > POPC-lipid A = POPC-POPG > POPC. Low peptide hydrophobicity and enhanced flexibility reduced binding. The influence of the other properties on the free energy of partitioning was low, but an enhanced hydrogen-bonding ability and dipole moment resulted in remarkable variations in the contribution of enthalpy and entropy. In the presence of rough and smooth LPS, the binding-modulating role of these parameters decreased. The highly differentiated activity pattern against E. coli was poorly reflected in peptide binding to LPS-containing membranes. However, stronger partitioning into POPC-smooth LPS than into POPC-rough LPS uncovered a significant role of O-antigen and outer core oligosaccharides in peptide transport and the permeabilization of the outer membrane and the anti-E. coli activity of the cyclic peptides.

Ca2+-activated Cl- currents are dispensable for olfaction
Billig, G. M., Pal, B., Fidzinski, P.; Jentsch, T. J.
Nat Neurosci, 14:763-769

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Canonical olfactory signal transduction involves the activation of cyclic AMP-activated cation channels that depolarize the cilia of receptor neurons and raise intracellular calcium. Calcium then activates Cl(-) currents that may be up to tenfold larger than cation currents and are believed to powerfully amplify the response. We identified Anoctamin2 (Ano2, also known as TMEM16B) as the ciliary Ca(2+)-activated Cl(-) channel of olfactory receptor neurons. Ano2 is expressed in the main olfactory epithelium (MOE) and in the vomeronasal organ (VNO), which also expresses the related Ano1 channel. Disruption of Ano2 in mice virtually abolished Ca(2+)-activated Cl(-) currents in the MOE and VNO. Ano2 disruption reduced fluid-phase electro-olfactogram responses by only approximately 40%, did not change air-phase electro-olfactograms and did not reduce performance in olfactory behavioral tasks. In contrast with the current view, cyclic nucleotide-gated cation channels do not need a boost by Cl(-) channels to achieve near-physiological levels of olfaction.

Occludin protein family: oxidative stress and reducing conditions
Blasig, I. E., Bellmann, C., Cording, J., Del Vecchio, G., Zwanziger, D., Huber(*), O.; Haseloff, R. F.
Antioxid Redox Signal, 15:1195-1219

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

Abstract: The occludin-like proteins belong to a family of tetraspan transmembrane proteins carrying a marvel domain. The intrinsic function of the occludin family is not yet clear. Occludin is a unique marker of any tight junction and is found in polarized endothelial and epithelial tissue barriers, at least in the adult vertebrate organism. Occludin is able to oligomerize and to form tight junction strands by homologous and heterologous interactions, but has no direct tightening function. Its oligomerization is affected by pro- and antioxidative agents or processes. Phosphorylation of occludin has been described at multiple sites and is proposed to play a regulatory role in tight junction assembly and maintenance and, hence, to influence tissue barrier characteristics. Redox-dependent signal transduction mechanisms are among the pathways modulating occludin phosphorylation and function. This review discusses the novel concept that occludin plays a key role in the redox regulation of tight junctions, which has a major impact in pathologies related to oxidative stress and corresponding pharmacologic interventions.

Identification of CYP106A2 as a Regioselective Allylic Bacterial Diterpene Hydroxylase
Bleif(*), S., Hannemann(*), F., Lisurek, M., von Kries, J. P., Zapp(*), J., Dietzen(*), M., Antes(*), I.; Bernhardt(*), R.
Chembiochem, 12:576-582

Tags: Screening Unit (von Kries), Computational Chemistry/ Drug Design (Kühne)

Abstract: The cytochrome P450 monooxygenase CYP106A2 from Bacillus megaterium ATCC 13368 catalyzes hydroxylations of a variety of 3-oxo-Delta(4)-steroids such as progesterone and deoxycorticosterone (DOC), mainly in the 15 beta-position. We combined a high-throughput screening and a rational approach for identifying new substrates of CYP106A2. The diterpene resin acid abietic acid was found to be a substrate and was docked into the active site of a CYP106A2 homology model to provide further inside into the structural basis of the regioselectivity of hydroxylation. The products of the hydroxylation reaction were analyzed by HPLC and the V(max) and K(m) values were calculated. The corresponding reaction products were analyzed by NMR spectroscopy and identified as 12 alpha- and 12 beta-hydroxyabietic acid. CYP106A2 was therefore identified as the first reported bacterial cytochrome P450 diterpene hydroxylase. Furthermore, an effective whole-cell catalyst for the selective allylic 12 alpha- and 12 beta-hydroxylation was applied to produce the hydroxylated products.

CIN85 Interacting Proteins in B Cells-Specific Role for SHIP-1
Büchse(*), T., Horras(*), N., Lenfert(*), E., Krystal(*), G., Korbel(*), S., Schümann, M., Krause, E., Mikkat(*), S.; Tiedge(*), M.
Mol. Cell. Proteomics, 10

Tags: Mass Spectrometry (Krause, E.)

Abstract: The Cbl-interacting 85-kDa protein (CIN85) plays an important role as a negative regulator of signaling pathways induced by receptor tyrosine kinases. By assembling multiprotein complexes this versatile adaptor enhances receptor tyrosine kinase-activated clathrin-mediated endocytosis and reduces phosphatidylinositol-3-kinase-induced phosphatidylinositol-3,4,5-trisphosphate production. Here we report the expression of CIN85 in primary splenic B lymphocytes and the B-lymphoma cell lines WEHI 231 and Ba/F3. Cross-linking of the B cell antigen receptor resulted in an increased association of CIN85 with the ubiquitin ligase Cbl. Through a systematic pull-down proteomics approach we identified 51 proteins that interact with CIN85 in B cells, including proteins not shown previously to be CIN85-associated. Among these proteins, the SH2-containing inositol phosphatase 1 (SHIP-1) co-precipitated with both the full-length CIN85 and each of its three SH3 domains. We also showed that this association is constitutive and depends on a region of 79 amino acids near the carboxyl terminus of SHIP-1, a region rich in potential SH3 domain binding sites. Because SHIP-1 is a major negative regulator of the phosphatidylinositol-3-kinase pathway in lymphocytes, we hypothesize that the interaction between SHIP-1 and CIN85 might synergistically facilitate the down-regulation of phosphatidylinositol-3,4,5-trisphosphate levels. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.006239, 1-12, 2011.

Small molecule AKAP-protein kinase A (PKA) interaction disruptors that activate PKA interfere with compartmentalized cAMP signaling in cardiac myocytes
Christian, F., Szaszak, M., Friedl, S., Drewianka(*), S., Lorenz, D., Goncalves, A., Furkert, J., Vargas, C., Schmieder, P., Götz, F., Zühlke, K., Moutty, M., Göttert, H., Joshi, M., Reif, B., Haase(*), H., Morano(*), I., Grossmann, S., Klukovits(*), A., Verli(*), J., Gaspar(*), R., Noack(*), C., Bergmann(*), M., Kass(*), R., Hampel(*), K., Kashin(*), D., Genieser(*), H. G., Herberg(*), F. W., Willoughby(*), D., Cooper(*), D. M., Baillie(*), G. S., Houslay(*), M. D., von Kries, J. P., Zimmermann(*), B., Rosenthal(*), W.; Klussmann, E.
J Biol Chem, 286:9079-9096

Tags: Anchored Signaling (Klussmann), Cellular Imaging (Wiesner), Solution NMR (Schmieder)

Abstract: A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating beta-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.

Spinophilin regulates central angiotensin II-mediated effect on blood pressure
da Costa Goncalves, A. C., Fontes(*), M. A., Klussmann(*), E., Qadri(*), F., Janke(*), J., Gollasch(*), M., Schleifenbaum(*), J., Müller(*), D., Jordan(*), J., Tank(*), J., Luft(*), F. C.; Gross(*), V.
J Mol Med (Berl), 89:1219-1229

Tags: Anchored Signaling (Klussmann)

Abstract: Central angiotensin II (AngII) plays an important role in the regulation of the sympathetic nervous system. The underlining molecular mechanisms are largely unknown. Spinophilin (SPL) is a regulator of G protein-coupled receptor signaling. Deletion of SPL induces sympathetically mediated arterial hypertension in mice. We tested the hypothesis that SPL restrains blood pressure (BP) by regulating AngII activity. We equipped SPL(-/-) and SPL(+/+) mice with telemetric devices and applied AngII (1.0 mg kg(-1) day(-1), minipumps) or the AngII subtype 1 receptor (AT1-R) blocker valsartan (50 mg kg(-1) day(-1), gavage). We assessed autonomic nervous system activity through intraperitoneal application of trimethaphan, metoprolol, and atropine. We also tested the effect of intracerebroventricular (icv) AngII on blood pressure in SPL(-/-) and in SPL(+/+) mice. Chronic infusion of AngII upregulates SPL expression in the hypothalamus of SPL(+/+) mice. Compared with SPL(+/+) mice, SPL(-/-) mice showed a greater increase in daytime BP with AngII (19.2 +/- 0.8 vs. 13.5 +/- 1.6 mmHg, p < 0.05). SPL(-/-) showed a greater depressor response to valsartan. BP and heart rate decreased more with trimethaphan and metoprolol in AngII-treated SPL(-/-) than in AngII-treated SPL(+/+) mice. SPL(-/-) mice responded more to icv AngII. Furthermore, brainstem AT1-R and AngII type 2 receptor (AT2-R) expression was reduced in SPL(-/-) mice. AngII treatment normalized AT1-R and AT2-R expression levels. In summary, our findings suggest that SPL restrains AngII-mediated sympathetic nervous system activation. SPL is a hitherto unrecognized molecule with regard to central blood pressure control and may pave the way to novel strategies for the treatment of hypertension.

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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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