FMP Publications

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

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References
Microsecond time scale mobility in a solid protein as studied by the 15N R(1rho) site-specific NMR relaxation rates
Krushelnitsky(*), A., Zinkevich(*), T., Reichert(*), D., Chevelkov, V.; Reif, B.
J Am Chem Soc, 132:11850-11853
(2010)

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: For the first time, we have demonstrated the site-resolved measurement of reliable (i.e., free of interfering effects) (15)N R(1rho) relaxation rates from a solid protein to extract dynamic information on the microsecond time scale. (15)N R(1rho) NMR relaxation rates were measured as a function of the residue number in a (15)N,(2)H-enriched (with 10-20% back-exchanged protons at labile sites) microcrystalline SH3 domain of chicken alpha-spectrin. The experiments were performed at different temperatures and at different spin-lock frequencies, which were realized by on- and off-resonance spin-lock irradiation. The results obtained indicate that the interfering spin-spin contribution to the R(1rho) rate in a perdeuterated protein is negligible even at low spin-lock fields, in contrast to the case for normal protonated samples. Through correlation plots, the R(1rho) rates were compared with previous data for the same protein characterizing different kinds of internal mobility.

Comparison of solid-state dipolar couplings and solution relaxation data provides insight into protein backbone dynamics
Chevelkov, V., Xue(*), Y., Linser, R., Skrynnikov(*), N. R.; Reif, B.
J Am Chem Soc, 132:5015-5017
(2010)

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: Analyses of solution (15)N relaxation data and solid-state (1)H(N)-(15)N dipolar couplings from a small globular protein, alpha-spectrin SH3 domain, produce a surprisingly similar pattern of order parameters. This result suggests that there is little or no ns-mus dynamics throughout most of the sequence and, in particular, in the structured portion of the backbone. At the same time, evidence of ns-mus motions is found in the flexible loops and termini. These findings, corroborated by the MD simulations of alpha-spectrin SH3 in a hydrated crystalline environment and in solution, are consistent with the picture of protein dynamics that has recently emerged from the solution studies employing residual dipolar couplings.

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.

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.

Intermolecular protein-RNA interactions revealed by 2D 31P-15N magic angle spinning solid-state NMR spectroscopy
Jehle(*), S., Falb(*), M., Kirkpatrick(*), J. P., Oschkinat, H., van Rossum, B. J., Althoff(*), G.; Carlomagno(*), T.
J Am Chem Soc, 132:3842-3846
(2010)

Tags: Protein Structure (Oschkinat)

Abstract: The structural investigation of large RNP complexes by X-ray crystallography can be a difficult task due to the flexibility of the RNA and of the protein-RNA interfaces, which may hinder crystallization. In these cases, NMR spectroscopy is an attractive alternative to crystallography, although the large size of typical RNP complexes may limit the applicability of solution NMR. Solid-state NMR spectroscopy, however, is not subject to any intrinsic limitations with respect to the size of the object under investigation, with restrictions imposed solely by the sensitivity of the instrumentation. In addition, it does not require large, well-ordered crystals and can therefore be applied to flexible, partially disordered complexes. Here we show for the first time that solid-state NMR spectroscopy can be used to probe intermolecular interactions at the protein-RNA interface in RNP complexes. Distances between the (15)N nuclei of the protein backbone and the (31)P nuclei of the RNA backbone can be measured in TEDOR experiments and used as restraints in structure calculations. The distance measurement is accurate, as proven for the test case of the L7Ae-box C/D RNA complex, for which a crystal structure is available. The results presented here reveal the as yet unexplored potential of solid-state NMR spectroscopy in the investigation of large RNP complexes.

Effects of ACE2 inhibition in the post-myocardial infarction heart
Kim(*), M. A., Yang(*), D., Kida(*), K., Molotkova(*), N., Yeo(*), S. J., Varki(*), N., Iwata(*), M., Dalton(*), N. D., Peterson(*), K. L., Siems, W. E., Walther(*), T., Cowling(*), R. T., Kjekshus(*), J.; Greenberg(*), B.
Journal of cardiac failure, 16:777-785
(2010)

Tags: Biochemical Neurobiology (Siems)

Abstract: BACKGROUND: There is evidence that angiotensin-converting enzyme 2 (ACE2) is cardioprotective. To assess this in the post-myocardial infarction (MI) heart, we treated adult male Sprague-Dawley rats with either placebo (PL) or C16, a selective ACE2 inhibitor, after permanent coronary artery ligation or sham operation. METHODS AND RESULTS: Coronary artery ligation resulting in MI between 25% to 50% of the left ventricular (LV) circumference caused substantial cardiac remodeling. Daily C16 administration from postoperative days 2 to 28 at a dose that inhibited myocardial ACE2 activity was associated with a significant increase in MI size and reduction in LV % fractional shortening. Treatment with C16 did not significantly affect post-MI increases in LV end-diastolic dimension but did inhibit increases in wall thickness and fibrosis in non-infarcted LV. On postoperative day 7, C16 had no significant effect on the increased level of apoptosis in the infarct and border zones nor did it significantly affect capillary density surrounding the MI. It did, however, significantly reduce the number of c-kit(+) cells in the border region. CONCLUSIONS: These findings support the notion that ACE2 exerts cardioprotective effects by preserving jeopardized cardiomyocytes in the border zone. The reduction in hypertrophy and fibrosis with C16, however, suggests that ACE2 activity has diverse effects on post-MI remodeling.

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.

No evidence for a role of CLCN2 variants in idiopathic generalized epilepsy
Niemeyer(*), M. I., Cid(*), L. P., Sepulveda(*), F. V., Blanz(*), J., Auberson(*), M.; Jentsch, T. J.
Nat Genet, 42:3-3
(2010)

Tags: Physiology and Pathology of Ion Transport (Jentsch

Participation of the second extracellular loop of claudin-5 in paracellular tightening against ions, small and large molecules
Piehl, C., Piontek, J., Cording, J., Wolburg(*), H.; Blasig, I. E.
Cellular and molecular life sciences : CMLS, 67:2131-2140
(2010)

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

Abstract: Tight junctions control paracellular permeability. Here, we analyzed the impact of residues in the second extracellular loop (ECL2) of mouse claudin-5 on paracellular permeability. Stable expression of claudin-5(wild type) in MDCK-II cells-but not that of mutants R145A, Y148A, Y158A or E159Q-increased transepithelial electrical resistance and decreased fluorescein permeation. Expression of claudin-5(Y148A), (Y158A) or (E159Q) enhanced permeability of FITC-dextran(10 kDa), which was unchanged in cells expressing claudin-5(wild type) or claudin-5(R145A). In contrast, targeting to tight junctions, strand morphology and tight junction assembly were unchanged. It is concluded that R145 is unessential for trans-interaction of claudin-5, but necessary for tightening against small solutes and ions. The highly conserved residues Y148, Y158 and E159 in ECL2 of claudin-5 contribute to homo- and/or heterophilic trans-interaction between classic claudins and thereby tighten the paracellular space against ions, small and large molecules. These results provide novel insights into the molecular function of tight junctions.

Distinct Neuropathologic Phenotypes After Disrupting the Chloride Transport Proteins ClC-6 or ClC-7/Ostm1
Pressey(*), S. N. R., O'Donnell(*), K. J., Stauber, T., Fuhrmann, J. C., Tyynela(*), J., Jentsch, T. J.; Cooper(*), J. D.
J Neuropath Exp Neur, 69:1228-1246
(2010)

Tags: Physiology and Pathology of Ion Transport (Jentsch

Abstract: The proteins ClC-6 and ClC-7 are expressed in the endosomallysosomal system. Because Clcn6-deficient mice display some features of neuronal ceroid lipofuscinosis (NCL), CLCN6 may be a candidate gene for novel forms of NCL. Using landmarks of disease progression from NCL mouse models as a guide, we examined neuropathologic alterations in the central nervous system of Clcn6(-/-), Clcn7(-/-), and gl mice. gl mice bear a mutation in Ostm1, the beta-subunit critical for Clcn7 function. Severely affected Clcn7(-/-) and gl mice have remarkably similar neuropathologic phenotypes, with pronounced reactive changes and neuron loss in the thalamocortical system, similar to findings in early-onset forms of NCL. In contrast, Clcn6(-/-) mice display slowly progressive, milder neuropathologic features with very little thalamic involvement or microglial activation. These findings detail for the first time the markedly different neuropathologic consequences of mutations in these two CLC genes. Clcn7(-/-) and gl mice bear a close resemblance to the progressive neuropathologic phenotypes of early onset forms of NCL, whereas the distinct phenotype of Clcn6-deficient mice suggests that this gene could be a candidate for a later-onset form of mild neurologic dysfunction with some NCL-like features.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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13125 Berlin, Germany
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info(at)fmp-berlin.de

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