FMP Publications

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

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References
Structural basis for the dissociation of alpha-synuclein fibrils triggered by pressure perturbation of the hydrophobic core
de Oliveira(*), G. A., Marques(*), M. A., Cruzeiro-Silva(*), C., Cordeiro(*), Y., Schuabb(*), C., Moraes(*), A. H., Winter(*), R., Oschkinat, H., Foguel(*), D., Freitas(*), M. S.; Silva(*), J. L.
Sci Rep, 6:37990
(2016)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Parkinson's disease is a neurological disease in which aggregated forms of the alpha-synuclein (alpha-syn) protein are found. We used high hydrostatic pressure (HHP) coupled with NMR spectroscopy to study the dissociation of alpha-syn fibril into monomers and evaluate their structural and dynamic properties. Different dynamic properties in the non-amyloid-beta component (NAC), which constitutes the Greek-key hydrophobic core, and in the acidic C-terminal region of the protein were identified by HHP NMR spectroscopy. In addition, solid-state NMR revealed subtle differences in the HHP-disturbed fibril core, providing clues to how these species contribute to seeding alpha-syn aggregation. These findings show how pressure can populate so far undetected alpha-syn species, and they lay out a roadmap for fibril dissociation via pathways not previously observed using other approaches. Pressure perturbs the cavity-prone hydrophobic core of the fibrils by pushing water inward, thereby inducing the dissociation into monomers. Our study offers the molecular details of how hydrophobic interaction and the formation of water-excluded cavities jointly contribute to the assembly and stabilization of the fibrils. Understanding the molecular forces behind the formation of pathogenic fibrils uncovered by pressure perturbation will aid in the development of new therapeutics against Parkinson's disease.

Insight into the Modification of Polymeric Micellar and Liposomal Nanocarriers by Fluorescein-Labeled Lipids and Uptake-Mediating Lipopeptides
Draffehn(*), S., Eichhorst, J., Wiesner, B.; Kumke(*), M. U.
Langmuir, 32:6928-6939
(2016)

Tags: Cellular Imaging (Wiesner)

Abstract: Encapsulation of diagnostic and therapeutic compounds in transporters improves their delivery to the point of need. An even more efficient treatment of diseases can be achieved using carriers with targeting or protecting moieties. In the present work, we investigated micellar and liposomal nanocarriers modified with fluorescein, peptides, and polymers that are covalently bound to fatty acids or phospholipids to ensure a self-driven incorporation into the micelles or liposomes. First, we characterized the photophysics of the fluorescent probes in the absence and in the presence of nanocarriers. Changes in the fluorescence decay time, quantum yield, and intensity of a fluorescein-labeled fatty acid (fluorescein-labeled palmitic acid [fPA]) and a fluorescein-labeled lipopeptide (P2fA2) were found. By exploiting these changes, we investigated a lipopeptide (P2A2 as an uptake-mediating unit) in combination with different nanocarriers (micelles and liposomes) and determined the corresponding association constant Kass values, which were found to be very high. In addition, the mobility of fPA was exploited using fluorescence correlation spectroscopy (FCS) and fluorescence depolarization (FD) experiments to characterize the nanocarriers. Cellular uptake experiments with mouse brain endothelial cells provided information on the uptake behavior of liposomes modified by uptake-mediating P2A2 and revealed differences in the uptake behavior between pH-sensitive and pH-insensitive liposomes.

Bimodal antagonism of PKA signalling by ARHGAP36
Eccles(*), R. L., Czajkowski(*), M. T., Barth(*), C., Müller(*), P. M., McShane(*), E., Grunwald(*), S., Beaudette(*), P., Mecklenburg(*), N., Volkmer, R., Zühlke(*), K., Dittmar(*), G., Selbach(*), M., Hammes(*), A., Daumke(*), O., Klussmann(*), E., Urbe(*), S.; Rocks(*), O.
Nat Commun, 7:12963
(2016)

Tags: Peptide Synthesis (Hackenberger/Volkmer)

Abstract: Protein kinase A is a key mediator of cAMP signalling downstream of G-protein-coupled receptors, a signalling pathway conserved in all eukaryotes. cAMP binding to the regulatory subunits (PKAR) relieves their inhibition of the catalytic subunits (PKAC). Here we report that ARHGAP36 combines two distinct inhibitory mechanisms to antagonise PKA signalling. First, it blocks PKAC activity via a pseudosubstrate motif, akin to the mechanism employed by the protein kinase inhibitor proteins. Second, it targets PKAC for rapid ubiquitin-mediated lysosomal degradation, a pathway usually reserved for transmembrane receptors. ARHGAP36 thus dampens the sensitivity of cells to cAMP. We show that PKA inhibition by ARHGAP36 promotes derepression of the Hedgehog signalling pathway, thereby providing a simple rationale for the upregulation of ARHGAP36 in medulloblastoma. Our work reveals a new layer of PKA regulation that may play an important role in development and disease.

A Small-Molecule Antagonist of the beta-Catenin/TCF4 Interaction Blocks the Self-Renewal of Cancer Stem Cells and Suppresses Tumorigenesis
Fang(*), L., Zhu(*), Q., Neuenschwander, M., Specker, E., Wulf-Goldenberg(*), A., Weis(*), W. I., von Kries, J. P.; Birchmeier(*), W.
Cancer research, 76:891-901
(2016)

Tags: Screening Unit (von Kries)

Abstract: Wnt/beta-catenin signaling is a highly conserved pathway essential for embryogenesis and tissue homeostasis. However, deregulation of this pathway can initiate and promote human malignancies, especially of the colon and head and neck. Therefore, Wnt/beta-catenin signaling represents an attractive target for cancer therapy. We performed high-throughput screening using AlphaScreen and ELISA techniques to identify small molecules that disrupt the critical interaction between beta-catenin and the transcription factor TCF4 required for signal transduction. We found that compound LF3, a 4-thioureido-benzenesulfonamide derivative, robustly inhibited this interaction. Biochemical assays revealed clues that the core structure of LF3 was essential for inhibition. LF3 inhibited Wnt/beta-catenin signals in cells with exogenous reporters and in colon cancer cells with endogenously high Wnt activity. LF3 also suppressed features of cancer cells related to Wnt signaling, including high cell motility, cell-cycle progression, and the overexpression of Wnt target genes. However, LF3 did not cause cell death or interfere with cadherin-mediated cell-cell adhesion. Remarkably, the self-renewal capacity of cancer stem cells was blocked by LF3 in concentration-dependent manners, as examined by sphere formation of colon and head and neck cancer stem cells under nonadherent conditions. Finally, LF3 reduced tumor growth and induced differentiation in a mouse xenograft model of colon cancer. Collectively, our results strongly suggest that LF3 is a specific inhibitor of canonical Wnt signaling with anticancer activity that warrants further development for preclinical and clinical studies as a novel cancer therapy.

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.

Collapse of redox homeostasis during aging and stress
Feleciano, D. R.; Kirstein, J.
Mol Cell Oncol, 3:e1091060
(2016)

Tags: Proteostasis in Aging and Disease (Kirstein)

Abstract: Coordination of the protein homeostasis network and redox states in eukaryotic cells is crucial for cellular and organismal fitness. By employing endogenous in vivo redox sensors we demonstrate that the redox state of the ER and cytosol is subject to profound changes upon proteotoxic challenges and during aging.

High resolution observed in 800 MHz DNP spectra of extremely rigid type III secretion needles
Fricke, P., Mance(*), D., Chevelkov, V., Giller(*), K., Becker(*), S., Baldus(*), M.; Lange, A.
J Biomol NMR, 65:121-126
(2016)

Tags: Molecular Biophysics (Lange, A.)

Abstract: The cryogenic temperatures at which dynamic nuclear polarization (DNP) solid-state NMR experiments need to be carried out cause line-broadening, an effect that is especially detrimental for crowded protein spectra. By increasing the magnetic field strength from 600 to 800 MHz, the resolution of DNP spectra of type III secretion needles (T3SS) could be improved by 22 %, indicating that inhomogeneous broadening is not the dominant effect that limits the resolution of T3SS needles under DNP conditions. The outstanding spectral resolution of this system under DNP conditions can be attributed to its low overall flexibility.

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).

Identification of Novel Nuclear Factor of Activated T Cell (NFAT)-associated Proteins in T Cells
Gabriel(*), C. H., Gross(*), F., Karl(*), M., Stephanowitz, H., Hennig(*), A. F., Weber(*), M., Gryzik(*), S., Bachmann(*), I., Hecklau(*), K., Wienands(*), J., Schuchhardt(*), J., Herzel(*), H., Radbruch(*), A., Krause, E.; Baumgrass(*), R.
J Biol Chem, 291:24172-24187
(2016)

Tags: Mass Spectrometry (Krause, E.)

Abstract: Transcription factors of the nuclear factor of activated T cell (NFAT) family are essential for antigen-specific T cell activation and differentiation. Their cooperative DNA binding with other transcription factors, such as AP1 proteins (FOS, JUN, and JUNB), FOXP3, IRFs, and EGR1, dictates the gene regulatory action of NFATs. To identify as yet unknown interaction partners of NFAT, we purified biotin-tagged NFATc1/alphaA, NFATc1/betaC, and NFATc2/C protein complexes and analyzed their components by stable isotope labeling by amino acids in cell culture-based mass spectrometry. We revealed more than 170 NFAT-associated proteins, half of which are involved in transcriptional regulation. Among them are many hitherto unknown interaction partners of NFATc1 and NFATc2 in T cells, such as Raptor, CHEK1, CREB1, RUNX1, SATB1, Ikaros, and Helios. The association of NFATc2 with several other transcription factors is DNA-dependent, indicating cooperative DNA binding. Moreover, our computational analysis discovered that binding motifs for RUNX and CREB1 are found preferentially in the direct vicinity of NFAT-binding motifs and in a distinct orientation to them. Furthermore, we provide evidence that mTOR and CHEK1 kinase activity influence NFAT's transcriptional potency. Finally, our dataset of NFAT-associated proteins provides a good basis to further study NFAT's diverse functions and how these are modulated due to the interplay of multiple interaction partners.

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.

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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
+4930 94793 - 100 
+4930 94793 - 109 (Fax)
info(at)fmp-berlin.de

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