FMP Publications

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

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References
Cellular Cations Control Conformational Switching of Inositol Pyrophosphate Analogues
Hager(*), A., Wu(*), M., Wang(*), H., Brown, N. W., Jr., Shears(*), S. B., Veiga(*), N.; Fiedler, D.
Chemistry, 22:12406-12414
(2016)

Tags: Chemical Biology I (Fiedler)

Abstract: The inositol pyrophosphate messengers (PP-InsPs) are emerging as an important class of cellular regulators. These molecules have been linked to numerous biological processes, including insulin secretion and cancer cell migration, but how they trigger such a wide range of cellular responses has remained unanswered in many cases. Here, we show that the PP-InsPs exhibit complex speciation behaviour and propose that a unique conformational switching mechanism could contribute to their multifunctional effects. We synthesised non-hydrolysable bisphosphonate analogues and crystallised the analogues in complex with mammalian PPIP5K2 kinase. Subsequently, the bisphosphonate analogues were used to investigate the protonation sequence, metal-coordination properties, and conformation in solution. Remarkably, the presence of potassium and magnesium ions enabled the analogues to adopt two different conformations near physiological pH. Understanding how the intrinsic chemical properties of the PP-InsPs can contribute to their complex signalling outputs will be essential to elucidate their regulatory functions.

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.

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.

Reversible Opening of Intercellular Junctions of Intestinal Epithelial and Brain Endothelial Cells With Tight Junction Modulator Peptides
Bocsik(*), A., Walter(*), F. R., Gyebrovszki(*), A., Fulop(*), L., Blasig, I., Dabrowski, S., Otvos(*), F., Toth(*), A., Rakhely(*), G., Veszelka(*), S., Vastag(*), M., Szabo-Revesz(*), P.; Deli(*), M. A.
Journal of pharmaceutical sciences, 105:754-765
(2016)

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

Abstract: The intercellular junctions restrict the free passage of hydrophilic compounds through the paracellular clefts. Reversible opening of the tight junctions of biological barriers is investigated as one of the ways to increase drug delivery to the systemic circulation or the central nervous system. Six peptides, ADT-6, HAV-6, C-CPE, 7-mer (FDFWITP, PN-78), AT-1002, and PN-159, acting on different integral membrane and linker junctional proteins were tested on Caco-2 intestinal epithelial cell line and a coculture model of the blood-brain barrier. All peptides tested in nontoxic concentrations showed a reversible tight junctions modulating effect and were effective to open the paracellular pathway for the marker molecules fluorescein and albumin. The change in the structure of cell-cell junctions was verified by immunostaining for occludin, claudin-4,-5, ZO-1, beta-catenin, and E-cadherin. Expression levels of occludin and claudins were measured in both models. We could demonstrate a selectivity of C-CPE, ADT-6, and HAV-6 peptides for epithelial cells and 7-mer and AT-1002 peptides for brain endothelial cells. PN-159 was the most effective modulator of junctional permeability in both models possibly acting via claudin-1 and -5. Our results indicate that these peptides can be effectively and selectively used as potential pharmaceutical excipients to improve drug delivery across biological barriers.

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.

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.

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.

Inositol polyphosphates intersect with signaling and metabolic networks via two distinct mechanisms
Wu(*), M., Chong, L. S., Perlman(*), D. H., Resnick(*), A. C.; Fiedler, D.
Proc Natl Acad Sci U S A, 113:E6757-E6765
(2016)

Tags: Chemical Biology I (Fiedler)

Abstract: Inositol-based signaling molecules are central eukaryotic messengers and include the highly phosphorylated, diffusible inositol polyphosphates (InsPs) and inositol pyrophosphates (PP-InsPs). Despite the essential cellular regulatory functions of InsPs and PP-InsPs (including telomere maintenance, phosphate sensing, cell migration, and insulin secretion), the majority of their protein targets remain unknown. Here, the development of InsP and PP-InsP affinity reagents is described to comprehensively annotate the interactome of these messenger molecules. By using the reagents as bait, >150 putative protein targets were discovered from a eukaryotic cell lysate (Saccharomyces cerevisiae). Gene Ontology analysis of the binding partners revealed a significant overrepresentation of proteins involved in nucleotide metabolism, glucose metabolism, ribosome biogenesis, and phosphorylation-based signal transduction pathways. Notably, we isolated and characterized additional substrates of protein pyrophosphorylation, a unique posttranslational modification mediated by the PP-InsPs. Our findings not only demonstrate that the PP-InsPs provide a central line of communication between signaling and metabolic networks, but also highlight the unusual ability of these molecules to access two distinct modes of action.

In vivo evaluation of riboflavin receptor targeted fluorescent USPIO in mice with prostate cancer xenografts
Jayapaul, J., Arns(*), S., Bunker(*), M., Weiler(*), M., Rutherford(*), S., Comba(*), P.; Kiessling(*), F.
Nano Res, 9:1319-1333
(2016)

Tags: Molecular Imaging (Schröder)

Abstract: Riboflavin (Rf) receptors bind and translocate Rf and its phosphorylated forms (e.g. flavin mononucleotide, FMN) into cells where they mediate various cellular metabolic pathways. Previously, we showed that FMN-coated ultrasmall superparamagnetic iron oxide (FLUSPIO) nanoparticles are suitable for labeling metabolically active cancer and endothelial cells in vitro. In this study, we focused on the in vivo application of FLUSPIO using prostate cancer xenografts. Size, charge, and chemical composition of FLUSPIO were evaluated. We explored the in vitro specificity of FLUSPIO for its cellular receptors using magnetic resonance imaging (MRI) and Prussian blue staining. Competitive binding experiments were performed in vivo by injecting free FMN in excess. Bio-distribution of FLUSPIO was determined by estimating iron content in organs and tumors using a colorimetric assay. AFM analysis and zeta potential measurements revealed a particulate morphology approximately 20-40 nm in size and a negative zeta potential (-24.23 +/- 0.15 mV) in water. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry data confirmed FMN present on the USPIO nanoparticle surface. FLUSPIO uptake in prostate cancer cells and human umbilical vein endothelial cells was significantly higher than that of control USPIO, while addition of excess of free FMN reduced accumulation. Similarly, in vivo MRI and histology showed specific FLUSPIO uptake by prostate cancer cells, tumor endothelial cells, and tumor-associated macrophages. Besides prominent tumor accumulation, FLUSPIO accumulated in the liver, spleen, lung, and skin. Hence, our data strengthen our hypothesis that targeting riboflavin receptors is an efficient approach to accumulate nanomedicines in tumors opening perspectives for the development of diagnostic and therapeutic systems. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available for this article at 10.1007/s12274-016-1028-7 and is accessible for authorized users.

Type II PI4-kinases control Weibel-Palade body biogenesis and von Willebrand factor structure in human endothelial cells
Lopes da Silva(*), M., O'Connor(*), M. N., Kriston-Vizi(*), J., White(*), I. J., Al-Shawi(*), R., Simons(*), J. P., Mössinger, J., Haucke, V.; Cutler(*), D. F.
J Cell Sci, 129:2096-2105
(2016)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Weibel-Palade bodies (WPBs) are endothelial storage organelles that mediate the release of molecules involved in thrombosis, inflammation and angiogenesis, including the pro-thrombotic glycoprotein von Willebrand factor (VWF). Although many protein components required for WPB formation and function have been identified, the role of lipids is almost unknown. We examined two key phosphatidylinositol kinases that control phosphatidylinositol 4-phosphate levels at the trans-Golgi network, the site of WPB biogenesis. RNA interference of the type II phosphatidylinositol 4-kinases PI4KIIalpha and PI4KIIbeta in primary human endothelial cells leads to formation of an increased proportion of short WPB with perturbed packing of VWF, as exemplified by increased exposure of antibody-binding sites. When stimulated with histamine, these cells release normal levels of VWF yet, under flow, form very few platelet-catching VWF strings. In PI4KIIalpha-deficient mice, immuno-microscopy revealed that VWF packaging is also perturbed and these mice exhibit increased blood loss after tail cut compared to controls. This is the first demonstration that lipid kinases can control the biosynthesis of VWF and the formation of WPBs that are capable of full haemostatic function.

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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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