FMP Publications

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

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References
On The Potential of Dynamic Nuclear Polarization Enhanced Diamonds in Solid-State and Dissolution (13) C NMR Spectroscopy
Bretschneider(*), C. O., Akbey, Ü., Aussenac(*), F., Olsen(*), G. L., Feintuch(*), A., Oschkinat, H.; Frydman(*), L.
Chemphyschem, 17:2691-2701
(2016)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Dynamic nuclear polarization (DNP) is a versatile option to improve the sensitivity of NMR and MRI. This versatility has elicited interest for overcoming potential limitations of these techniques, including the achievement of solid-state polarization enhancement at ambient conditions, and the maximization of (13) C signal lifetimes for performing in vivo MRI scans. This study explores whether diamond's (13) C behavior in nano- and micro-particles could be used to achieve these ends. The characteristics of diamond's DNP enhancement were analyzed for different magnetic fields, grain sizes, and sample environments ranging from cryogenic to ambient temperatures, in both solution and solid-state experiments. It was found that (13) C NMR signals could be boosted by orders of magnitude in either low- or room-temperature solid-state DNP experiments by utilizing naturally occurring paramagnetic P1 substitutional nitrogen defects. We attribute this behavior to the unusually long electronic/nuclear spin-lattice relaxation times characteristic of diamond, coupled with a time-independent cross-effect-like polarization transfer mechanism facilitated by a matching of the nitrogen-related hyperfine coupling and the (13) C Zeeman splitting. The efficiency of this solid-state polarization process, however, is harder to exploit in dissolution DNP-enhanced MRI contexts. The prospects for utilizing polarized diamond approaching nanoscale dimensions for both solid and solution applications are briefly discussed.

Temperature dependence of cross-effect dynamic nuclear polarization in rotating solids: advantages of elevated temperatures
Geiger, M. A., Orwick-Rydmark, M., Marker, K., Franks, W. T., Akhmetzyanov(*), D., Stöppler, D., Zinke, M., Specker, E., Nazare, M., Diehl, A., van Rossum, B. J., Aussenac(*), F., Prisner(*), T., Akbey, Ü.; Oschkinat, H.
Phys Chem Chem Phys, 18:30696-30704
(2016)

Tags: NMR-Supported Structural Biology (Oschkinat), Medicinal Chemistry (Nazare), Molecular Biophysics (Lange, A.)

Abstract: Dynamic nuclear polarization exploits electron spin polarization to boost signal-to-noise in magic-angle-spinning (MAS) NMR, creating new opportunities in materials science, structural biology, and metabolomics studies. Since protein NMR spectra recorded under DNP conditions can show improved spectral resolution at 180-200 K compared to 110 K, we investigate the effects of AMUPol and various deuterated TOTAPOL isotopologues on sensitivity and spectral resolution at these temperatures, using proline and reproducibly prepared SH3 domain samples. The TOTAPOL deuteration pattern is optimized for protein DNP MAS NMR, and signal-to-noise per unit time measurements demonstrate the high value of TOTAPOL isotopologues for Protein DNP MAS NMR at 180-200 K. The combined effects of enhancement, depolarization, and proton longitudinal relaxation are surprisingly sample-specific. At 200 K, DNP on SH3 domain standard samples yields a 15-fold increase in signal-to-noise over a sample without radicals. 2D and 3D NCACX/NCOCX spectra were recorded at 200 K within 1 and 13 hours, respectively. Decreasing enhancements with increasing 2H-content at the CH2 sites of the TEMPO rings in CD3-TOTAPOL highlight the importance of protons in a sphere of 4-6 A around the nitroxyl group, presumably for polarization pickup from electron spins.

Studying the Conformation of a Silaffin-Derived Pentalysine Peptide Embedded in Bioinspired Silica using Solution and Dynamic Nuclear Polarization Magic-Angle Spinning NMR
Geiger(*), Y., Gottlieb(*), H. E., Akbey, Ü., Oschkinat, H.; Goobes(*), G.
J Am Chem Soc, 138:5561-5567
(2016)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Smart materials are created in nature at interfaces between biomolecules and solid materials. The ability to probe the structure of functional peptides that engineer biogenic materials at this heterogeneous setting can be facilitated tremendously by use of DNP-enhanced solid-state NMR spectroscopy. This sensitive NMR technique allows simple and quick measurements, often without the need for isotope enrichment. Here, it is used to characterize a pentalysine peptide, derived from a diatom's silaffin protein. The peptide accelerates the formation of bioinspired silica and gets embedded inside the material as it is formed. Two-dimensional DNP MAS NMR of the silica-bound peptide and solution NMR of the free peptide are used to derive its secondary structure in the two states and to pinpoint some subtle conformational changes that the peptide undergoes in order to adapt to the silica environment. In addition, interactions between abundant lysine residues and silica surface are identified, and proximity of other side chains to silica and to neighboring peptide molecules is discussed.

Interplay between redox and protein homeostasis
Feleciano, D. R., Arnsburg, K.; Kirstein, J.
Worm, 5:e1170273
(2016)

Tags: Proteostasis in Aging and Disease (Kirstein)

Abstract: The subcellular compartments of eukaryotic cells are characterized by different redox environments. Whereas the cytosol, nucleus and mitochondria are more reducing, the endoplasmic reticulum represents a more oxidizing environment. As the redox level controls the formation of intra- and inter-molecular disulfide bonds, the folding of proteins is tightly linked to its environment. The proteostasis network of each compartment needs to be adapted to the compartmental redox properties. In addition to chaperones, also members of the thioredoxin superfamily can influence the folding of proteins by regulation of cysteine reduction/oxidation. This review will focus on thioredoxin superfamily members and chaperones of C. elegans, which play an important role at the interface between redox and protein homeostasis. Additionally, this review will highlight recent methodological developments on in vivo and in vitro assessment of the redox state and their application to provide insights into the high complexity of redox and proteostasis networks of C. elegans.

Structural biology applications of solid state MAS DNP NMR
Akbey(*), Ü.; Oschkinat, H.
J Magn Reson, 269:213-224
(2016)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Dynamic Nuclear Polarization (DNP) has long been an aim for increasing sensitivity of nuclear magnetic resonance (NMR) spectroscopy, delivering spectra in shorter experiment times or of smaller sample amounts. In recent years, it has been applied in magic angle spinning (MAS) solid-state NMR to a large range of samples, including biological macromolecules and functional materials. New research directions in structural biology can be envisaged by DNP, facilitating investigations on very large complexes or very heterogeneous samples. Here we present a summary of state of the art DNP MAS NMR spectroscopy and its applications to structural biology, discussing the technical challenges and factors affecting DNP performance.

Progesterone, estrogen, and androgen receptors in the corpus luteum of the domestic cat, Iberian lynx (Lynx pardinus) and Eurasian lynx (Lynx lynx)
Amelkina(*), O., Zschockelt(*), L., Painer(*), J., Serra(*), R., Villaespesa(*), F., Krause, E., Jewgenow(*), K.; Braun(*), B. C.
Theriogenology, 86:2107-2118
(2016)

Tags: Mass Spectrometry (Krause, E.)

Abstract: In contrast to the species studied, the corpus luteum (CL) of Iberian and Eurasian lynx physiologically persists in the ovary for more than 2 years and continues to secrete progesterone. Such persistent CL (perCL) transition into the next cycle and are present in the ovary together with the freshly formed CL (frCL) of a new ovulation. To date, the mechanisms supporting such CL persistence are not known. We analyzed the potential receptivity of feline CL to sex steroids through mRNA measurements of progesterone receptor (PGR), progesterone receptor membrane components (PGRMC) 1 and 2, estrogen receptor (ESR) 1 and ESR2, G protein-coupled estrogen receptor 1 (GPER1), and androgen receptor (AR). All receptors were present in domestic cat CL during pregnancy and the nonpregnant luteal phase, in frCL and perCL of post-mating Iberian lynx and in perCL of pre-mating Eurasian lynx. Mass spectrometry detected the presence of PGRMC1 protein in frCL and perCL of the Iberian lynx. In both domestic cat and lynx CL, PGR, PGRMC1, and ESR1 proteins were localized in luteal cells by immunohistochemistry. The mRNA levels of PGR, PGRMC1, PGRMC2, ESR1, and AR changed significantly throughout the domestic cat luteal phase. This may indicate involvement of these receptors in the processes of formation, maintenance, and regression of feline CL. In Iberian lynx, expression of PGRMC1, PGRCM2, ESR1, GPER1, and AR was significantly higher in perCL compared with frCL, whereas ESR2 was reversed. High mRNA amounts of these receptors in perCL suggest that physiological persistence of lynx CL may be partly regulated by actions of sex steroids through their nuclear and/or membrane receptors.

Chemical fragment arrays for rapid druggability assessment
Aretz(*), J., Kondoh(*), Y., Honda(*), K., Anumala, U. R., Nazare, M., Watanabe(*), N., Osada(*), H.; Rademacher(*), C.
Chem Commun (Camb), 52:9067-9070
(2016)

Tags: Medicinal Chemistry (Nazare)

Abstract: Incorporation of early druggability assessment in the drug discovery process provides a means to prioritize target proteins for high-throughput screening. We present chemical fragment arrays as a method that is capable of determining the druggability of a given target with low protein and compound consumption, enabling rapid decision making during early phases of drug discovery.

Modulation of Hexadecyl-LPA-Mediated Activation of Mast Cells and Microglia by a Chemical Probe for LPA5
Kozian(*), D. H., von Haeften(*), E., Joho(*), S., Czechtizky(*), W., Anumala, U. R., Roux(*), P., Dudda(*), A., Evers(*), A.; Nazare, M.
Chembiochem, 17:861-865
(2016)

Tags: Medicinal Chemistry (Nazare)

Abstract: Mast cells and microglia play a critical role in innate immunity and inflammation and can be activated by a wide range of endogenous and exogenous stimuli. Lysophosphatidic acid (LPA) has recently been reported to activate mast cells and microglia. Using the human mast cell line HMC-1 and the mouse microglia cell line BV-2, we show that LPA-mediated activation can be prevented by blockade of the LPA receptor 5 (LPA5) in both cell lines. The identification of new LPA5-specific antagonists as tool compounds to probe and modulate the LPA5/LPA axis in relevant in vitro and in vivo assays should contribute to better understanding of the underlying role of LPAs in the development and progression of (neuro-) inflammatory diseases.

C-type natriuretic peptide and natriuretic peptide receptor B signalling inhibits cardiac sympathetic neurotransmission and autonomic function
Buttgereit(*), J., Shanks(*), J., Li(*), D., Hao(*), G., Athwal(*), A., Langenickel(*), T. H., Wright(*), H., da Costa Goncalves, A. C., Monti(*), J., Plehm(*), R., Popova(*), E., Qadri(*), F., Lapidus(*), I., Ryan(*), B., Ozcelik(*), C., Paterson(*), D. J., Bader(*), M.; Herring(*), N.
Cardiovasc Res, 112:637-644
(2016)

Tags: Anchored Signaling (Klussmann)

Abstract: AIMS: B-type natriuretic peptide (BNP)-natriuretic peptide receptor A (NPR-A) receptor signalling inhibits cardiac sympathetic neurotransmission, although C-type natriuretic peptide (CNP) is the predominant neuropeptide of the nervous system with expression in the heart and vasculature. We hypothesized that CNP acts similarly to BNP, and that transgenic rats (TGRs) with neuron-specific overexpression of a dominant negative NPR-B receptor would develop heightened sympathetic drive. METHODS AND RESULTS: Mean arterial pressure and heart rate (HR) were significantly (P < 0.05) elevated in freely moving TGRs (n = 9) compared with Sprague Dawley (SD) controls (n = 10). TGR had impaired left ventricular systolic function and spectral analysis of HR variability suggested a shift towards sympathoexcitation. Immunohistochemistry demonstrated co-staining of NPR-B with tyrosine hydroxylase in stellate ganglia neurons. In SD rats, CNP (250 nM, n = 8) significantly reduced the tachycardia during right stellate ganglion stimulation (1-7 Hz) in vitro whereas the response to bath-applied norepinephrine (NE, 1 muM, n = 6) remained intact. CNP (250 nM, n = 8) significantly reduced the release of 3H-NE in isolated atria and this was prevented by the NPR-B antagonist P19 (250 nM, n = 6). The neuronal Ca2+ current (n = 6) and intracellular Ca2+ transient (n = 9, using fura-2AM) were also reduced by CNP in isolated stellate neurons. Treatment of the TGR (n = 9) with the sympatholytic clonidine (125 microg/kg per day) significantly reduced mean arterial pressure and HR to levels observed in the SD (n = 9). CONCLUSION: C-type natriuretic peptide reduces cardiac sympathetic neurotransmission via a reduction in neuronal calcium signalling and NE release through the NPR-B receptor. Situations impairing CNP-NPR-B signalling lead to hypertension, tachycardia, and impaired left ventricular systolic function secondary to sympatho-excitation.

TNF induced cleavage of HSP90 by cathepsin D potentiates apoptotic cell death
Fritsch(*), J., Fickers(*), R., Klawitter(*), J., Särchen(*), V., Zingler(*), P., Adam(*), D., Janssen(*), O., Krause, E.; Schütze(*), S.
Oncotarget, 7:75774-75789
(2016)

Tags: Mass Spectrometry (Krause, E.)

Abstract: During apoptosis induction by TNF, the extrinsic and intrinsic apoptosis pathways converge at the lysosomal-mitochondrial interface. Earlier studies showed that the lysosomal aspartic protease Cathepsin D (CtsD) cleaves Bid to tBid, resulting in the amplification of the initial apoptotic cascade via mitochondrial outer membrane permeabilization (MOMP).The goal of this study was to identify further targets for CtsD that might be involved in activation upon death receptor ligation. Using a proteomics screen, we identified the heat shock protein 90 (HSP90) to be cleaved by CtsD after stimulation of U937 or other cell lines with TNF, FasL and TRAIL. HSP90 cleavage corresponded to apoptosis sensitivity of the cell lines to the different stimuli. After mutation of the cleavage site, HSP90 partially prevented apoptosis induction in U937 and Jurkat cells. Overexpression of the cleavage fragments in U937 and Jurkat cells showed no effect on apoptosis, excluding a direct pro-apoptotic function of these fragments. Pharmacological inhibition of HSP90 with 17AAG boosted ligand mediated apoptosis by enhancing Bid cleavage and caspase-9 activation. Together, we demonstrated that HSP90 plays an anti-apoptotic role in death receptor signalling and that CtsD-mediated cleavage of HSP90 sensitizes cells for apoptosis. These findings identify HSP90 as a potential target for cancer therapy in combination with death ligands (e.g. TNF or TRAIL).

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
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