FMP Publications

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

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References
The use of small molecule high-throughput screening to identify inhibitors of the proteinase 3-NB1 interaction
Choi(*), M., Eulenberg(*), C., Rolle(*), S., von Kries, J. P., Luft(*), F. C.; Kettritz(*), R.
Clin Exp Immunol, 161:389-396
(2010)

Tags: Screening Unit (von Kries)

Abstract: P>Anti-neutrophil cytoplasmic antibodies (ANCA) to proteinase 3 (PR3) are found in patients with small-vessel vasculitis. PR3-ANCA bind strongly to membrane PR3 (mPR3) that is presented by the NB1 receptor. We performed high-throughput screening using a small molecule library to identify compounds that inhibit PR3-NB1 binding. We established a human embryonic kidney (HEK293) cell-based system, where approximately 95 +/- 2% of the NB1-transfected cells expressed the NB1 receptor on the cell surface. Addition of 0 center dot 1 mu g/ml human PR3 to 104 NB1-expressing HEK293 cells resulted in PR3 binding that was detected by immunofluorescence using a fluorescence plate reader assay. We identified 13 of 20 000 molecules that inhibited PR3 binding by > 70%. Seven of 13 substances showed reproducible inhibition in four additional validation experiments. Two selected compounds (27519 and 27549) demonstrated a dose-dependent inhibition over a range from 6 center dot 25 to 100 mu M as measured by the plate reader assay. We used flow cytometry as a second assay, and found that both compounds reproducibly inhibited PR3 binding to NB1-transfected HEK293 cells at 50 mu M (inhibition to 42 +/- 4% with compound 27519 and to 47 +/- 6% with compound 27549 compared to the dimethylsulphoxide control). Furthermore, compounds 27519 and 27549 also inhibited binding of exogenous PR3 to human neutrophils. In contrast, the compounds did not decrease mPR3 expression on resting neutrophils, but reduced the tumour necrosis factor-alpha-mediated mPR3 increase on NB1pos neutrophils when present continuously during the assay. The findings suggest that small inhibitory compounds provide a potential therapeutic tool to reduce mPR3 by preventing its binding to NB1.

Efficient access to peptidyl ketones and peptidyl diketones via C-alkylations and C-acylations of polymer-supported phosphorus ylides followed by hydrolytic and/or oxidative cleavage
El-Dahshan, A., Ahsanullah, R.J.; Rademann, J.
Biopolymers, 94:220-228
(2010)

Tags: Medicinal Chemistry (Rademann)

Abstract: Novel syntheses of peptidyl ketones and peptidyl diketones on polymer support are described. Peptidyl phosphoranylidene acetates were prepared via C-acylation of polymer-supported phosphorus ylides. Selective alkylation of the ylide carbon with various alkyl halides, such as methyl iodide and benzyl bromide was established. Peptidyl diketones were obtained by oxidative cleavage. Peptidyl ketones were furnished by hydrolysis of the peptidyl phosphorus ylides under either basic or acidic conditions.

Short Cationic Antimicrobial Peptides Interact with ATP
Hilpert(*), K., McLeod(*), B., Yu(*), J., Elliott(*), M. R., Rautenbach(*), M., Ruden(*), S., Bürck(*), J., Muhle-Goll(*), C., Ulrich(*), A. S., Keller, S.; Hancock(*), R. E. W.
Antimicrob Agents Ch, 54:4480-4483
(2010)

Tags: Biophysics of Membrane Proteins (Keller)

Abstract: The mode of action of short, nonhelical antimicrobial peptides is still not well understood. Here we show that these peptides interact with ATP and directly inhibit the actions of certain ATP-dependent enzymes, such as firefly luciferase, DnaK, and DNA polymerase. alpha-Helical and planar or circular antimicrobial peptides did not show such interaction with ATP.

Signal transduction in CHO cells stably transfected with domain-selective forms of murine ACE
Sun, X., Rentzsch(*), B., Gong(*), M., Eichhorst, J., Pankow, K., Papsdorf, G., Maul, B., Bader(*), M.; Siems, W. E.
Biol Chem, 391:235-244
(2010)

Tags: Biochemical Neurobiology (Siems), Protein Trafficking (Schülein), Cellular Imaging (Wiesner)

Abstract: Membrane-bound human angiotensin-converting enzyme (ACE) has been reported to initiate intracellular signaling after interaction with substrates or inhibitors. Somatic ACE is known to contain two distinct, extracellular catalytic centers. We analyzed the signal transduction mechanisms in cells transfected with different forms of murine ACE (mACE) and investigated whether the two domains are similarly involved in these processes. For this purpose, CHO cells were stably transfected with mACE or with its domain-selective mutants. In addition to these modified cellular models, human umbilical vein endothelial cells were used in this study. Signal transduction molecules such as JNK and c-Jun were analyzed after activation of cells with several ACE substrates and inhibitors. ACE-targeting compounds such as substrates, inhibitors, or even the ACE product angiotensin-II induce in mACE-expressing cells a signal transduction response. These processes are also evoked by partially inactivated forms of mACE and finally result in an enhanced cyclooxygenase-2 transcription. Surprisingly, the membrane-bound ACE activity is also influenced by ACE-targeted interventions. Our data suggest that the two catalytic domains of mACE do not function independently but that the signal transduction is influenced by negative cooperativity of the two catalytic domains. This study underlines that ACE indeed has receptor-like properties which occur in a species-specific manner.

Structural basis of oligomerization in septin-like GTPase of immunity-associated protein 2 (GIMAP2)
Schwefel(*), D., Fröhlich(*), C., Eichhorst, J., Wiesner, B., Behlke(*), J., Aravind(*), L.; Daumke(*), O.
Proc Natl Acad Sci U S A, 107:20299-20304
(2010)

Tags: Cellular Imaging (Wiesner)

Abstract: GTPases of immunity-associated proteins (GIMAPs) are a distinctive family of GTPases, which control apoptosis in lymphocytes and play a central role in lymphocyte maturation and lymphocyte-associated diseases. To explore their function and mechanism, we determined crystal structures of a representative member, GIMAP2, in different nucleotide-loading and oligomerization states. Nucleotide-free and GDP-bound GIMAP2 were monomeric and revealed a guanine nucleotide-binding domain of the TRAFAC (translation factor associated) class with a unique amphipathic helix alpha7 packing against switch II. In the absence of alpha7 and the presence of GTP, GIMAP2 oligomerized via two distinct interfaces in the crystal. GTP-induced stabilization of switch I mediates dimerization across the nucleotide-binding site, which also involves the GIMAP specificity motif and the nucleotide base. Structural rearrangements in switch II appear to induce the release of alpha7 allowing oligomerization to proceed via a second interface. The unique architecture of the linear oligomer was confirmed by mutagenesis. Furthermore, we showed a function for the GIMAP2 oligomer at the surface of lipid droplets. Although earlier studies indicated that GIMAPs are related to the septins, the current structure also revealed a strikingly similar nucleotide coordination and dimerization mode as in the dynamin GTPase. Based on this, we reexamined the relationships of the septin- and dynamin-like GTPases and demonstrate that these are likely to have emerged from a common membrane-associated dimerizing ancestor. This ancestral property appears to be critical for the role of GIMAPs as nucleotide-regulated scaffolds on intracellular membranes.

Glycogen synthase kinase 3beta interaction protein functions as an A-kinase anchoring protein
Hundsrucker, C., Skroblin, P., Christian, F., Zenn(*), H. M., Popara, V., Joshi, M., Eichhorst, J., Wiesner, B., Herberg(*), F. W., Reif, B., Rosenthal(*), W.; Klussmann, E.
J Biol Chem, 285:5507-5521
(2010)

Tags: Anchored Signalling (Klussmann), Solid-State NMR Spectroscopy (Reif), Cellular Imaging (Wiesner)

Abstract: A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3beta interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3beta (glycogen synthase kinase 3beta). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3beta by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3beta and thereby provides a mechanism for the integration of PKA and GSK3beta signaling pathways.

A MAS NMR Study of the Bacterial ABC Transporter ArtMP
Lange, V., Becker-Baldus, J., Kunert, B., van Rossum, B. J., Casagrande(*), F., Engel(*), A., Roske(*), Y., Scheffel(*), F. M., Schneider(*), E.; Oschkinat, H.
Chembiochem, 11:547-555
(2010)

Tags: Protein Structure (Oschkinat)

Abstract: ATP-binding cassette (ABC) transport systems facilitate the translocation of substances, like amino acids, across cell membranes energised by ATP hydrolysis. This work describes first structural studies on the ABC transporter ArtMP from Geobacillus stearothermophilus in native lipid environment by magic-angle spinning NMR spectroscopy. The 2D crystals of ArtMP and 3D crystals of isolated ArtP were prepared in different nucleotide-bound or -unbound states. From selectively C-13,N-15-labelled ArtP, several sequence-specific assignments were obtained, most of which could be transferred to spectra of ArtMP. Residues Tyr133 and Pro134 protrude directly into the ATP-binding pocket at the interface of the ArtP subunits, and hence, are sensitive monitors for structural changes during nucleotide binding and hydrolysis. Distinct sets of NMR shifts were obtained for ArtP with different phosphorylation states of the ligand. Indications were found for an asymmetric or inhomogeneous state of the ArtP dimer bound with triphosphorylated nucleotides. With this investigation, a model system was established for screening all functional states occurring in one ABC transporter in native lipid environment.

Analysis of CLCN2 as Candidate Gene for Megalencephalic Leukoencephalopathy with Subcortical Cysts
Scheper(*), G. C., van Berkel(*), C. G. M., Leisle, L., de Groot(*), K. E., Errami(*), A., Jentsch, T. J.; Van der Knaap(*), M. S.
Genet Test Mol Bioma, 14:255-257
(2010)

Tags: Physiology and Pathology of Ion Transport (Jentsch

Abstract: Mutations in the gene MLC1 are found in approximately 80% of the patients with the inherited childhood white matter disorder megalencephalic leukoencephalopathy with subcortical cysts (MLC). Genetic linkage studies have not led to the identification of another disease gene. We questioned whether mutations in CLCN2, coding for the chloride channel protein 2 (ClC-2), are involved in MLC. Mice lacking this protein develop white matter abnormalities, which are characterized by vacuole formation in the myelin sheaths, strikingly similar to the intramyelinic vacuoles in MLC. Sequence analysis of CLCN2 at genomic DNA and cDNA levels in 18 MLC patients without MLC1 mutations revealed some nucleotide changes, but they were predicted to be nonpathogenic. Further, in electrophysiological experiments, one of the observed amino acid changes was shown to have no effect on the ClC-2-mediated currents. In conclusion, we found no evidence suggesting that the CLCN2 gene is involved in MLC.

Plasma ACE2 activity is an independent prognostic marker in Chagas' disease and equally potent as BNP
Wang(*), Y., Moreira Mda(*), C., Heringer-Walther(*), S., Ebermann(*), L., Schultheiss(*), H. P., Wessel(*), N., Siems, W. E.; Walther(*), T.
Journal of cardiac failure, 16:157-163
(2010)

Tags: Biochemical Neurobiology (Siems)

Abstract: BACKGROUND: Angiotensin-converting enzyme (ACE) 2 is a novel homologue of ACE. It metabolizes angiotensin (Ang)II to Ang-(1-7). This study aims to investigate the diagnostic and prognostic potency of circulating ACE2 activity in patients with heart failure (HF) from Chagas' disease (CD). METHODS AND RESULTS: Blood samples were obtained from 111 CD patients and 40 age- and gender-matched healthy subjects. The CD patients were further subdivided according to their New York Heart Association classification. ACE2 activity was significantly increased in CD patients with HF, but not in patients without systolic dysfunction. Moreover, plasma ACE2 activity was significantly correlated with their clinical severity and echocardiographic parameters. Importantly, the potency of circulating ACE2 activity in CD patients was equally potent as that of B-type natriuretic peptide to predict cardiac death and heart transplant. Most importantly, patients with both parameters elevated were on a 5-fold higher risk to reach an endpoint than patients with increase in only 1 of the 2 parameters. CONCLUSIONS: Determination of ACE2 activity may provide a new and important diagnostic and prognostic marker for patients with CD. ACE2 activity and BNP concentration have additive predictive value and may be used in combination to offer a new dimension of prediction in HF.

Effects of thawing, refreezing and storage conditions of tissue samples and protein extracts on 2-DE spot intensity
Weist(*), S., Brunkau(*), C., Wittke(*), J., Eravci(*), M., Broedel(*), O., Krause, E., Stephanowitz, H., Eravci(*), S.; Baumgartner(*), A.
Proteomics, 10:1515-1521
(2010)

Tags: Mass Spectrometry (Krause, E.)

Abstract: We report that reliable quantitative proteome analyses can be performed with tissue samples stored at -80 degrees C for up to 10 years. However, storing protein extracts at 4 degrees C for 24h and freezing protein extracts at -80 degrees C and thawing them significantly altered 41.6 and 17.5% of all spot intensities on 2-DE gels, respectively. Fortunately, these storing effects did not impair the reliability of quantifying 2-DE experiments. Nonetheless, the results show that freezing and storage conditions should be carefully controlled in proteomic experiments.

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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)
info(at)fmp-berlin.de

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