FMP Publications

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

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Preparation of biogenic gas vesicle nanostructures for use as contrast agents for ultrasound and MRI
Lakshmanan(*), A., Lu(*), G. J., Farhadi(*), A., Nety(*), S. P., Kunth, M., Lee-Gosselin(*), A., Maresca(*), D., Bourdeau(*), R. W., Yin(*), M., Yan(*), J., Witte, C., Malounda(*), D., Foster(*), F. S., Schröder, L.; Shapiro(*), M. G.
Nat Protoc, 12:2050-2080

Tags: Molecular Imaging (Schröder)

Abstract: Gas vesicles (GVs) are a unique class of gas-filled protein nanostructures that are detectable at subnanomolar concentrations and whose physical properties allow them to serve as highly sensitive imaging agents for ultrasound and MRI. Here we provide a protocol for isolating GVs from native and heterologous host organisms, functionalizing these nanostructures with moieties for targeting and fluorescence, characterizing their biophysical properties and imaging them using ultrasound and MRI. GVs can be isolated from natural cyanobacterial and haloarchaeal host organisms or from Escherichia coli expressing a heterologous GV gene cluster and purified using buoyancy-assisted techniques. They can then be modified by replacing surface-bound proteins with engineered, heterologously expressed variants or through chemical conjugation, resulting in altered mechanical, surface and targeting properties. Pressurized absorbance spectroscopy is used to characterize their mechanical properties, whereas dynamic light scattering (DLS)and transmission electron microscopy (TEM) are used to determine nanoparticle size and morphology, respectively. GVs can then be imaged with ultrasound in vitro and in vivo using pulse sequences optimized for their detection versus background. They can also be imaged with hyperpolarized xenon MRI using chemical exchange saturation transfer between GV-bound and dissolved xenon-a technique currently implemented in vitro. Taking 3-8 d to prepare, these genetically encodable nanostructures enable multimodal, noninvasive biological imaging with high sensitivity and potential for molecular targeting.

Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS
Leonhardt(*), J., Villela(*), D. C., Teichmann, A., Munter(*), L. M., Mayer(*), M. C., Mardahl(*), M., Kirsch(*), S., Namsolleck(*), P., Lucht(*), K., Benz(*), V., Alenina(*), N., Daniell(*), N., Horiuchi(*), M., Iwai(*), M., Multhaup(*), G., Schülein, R., Bader(*), M., Santos(*), R. A., Unger(*), T.; Steckelings(*), U. M.

Tags: Protein Trafficking (Schülein), Cellular Imaging (Wiesner)

Abstract: The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin-angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa. Such cross-inhibition may indicate heterodimerization of these receptors. Therefore, this study investigated the molecular and functional interplay between MAS and the AT2R. Molecular interactions were assessed by fluorescence resonance energy transfer and by cross correlation spectroscopy in human embryonic kidney-293 cells transfected with vectors encoding fluorophore-tagged MAS or AT2R. Functional interaction of AT2R and MAS was studied in astrocytes with CX3C chemokine receptor-1 messenger RNA expression as readout. Coexpression of fluorophore-tagged AT2R and MAS resulted in a fluorescence resonance energy transfer efficiency of 10.8 +/- 0.8%, indicating that AT2R and MAS are capable to form heterodimers. Heterodimerization was verified by competition experiments using untagged AT2R and MAS. Specificity of dimerization of AT2R and MAS was supported by lack of dimerization with the transient receptor potential cation channel, subfamily C-member 6. Dimerization of the AT2R was abolished when it was mutated at cysteine residue 35. AT2R and MAS stimulation with the respective agonists, Compound 21 or angiotensin-(1-7), significantly induced CX3C chemokine receptor-1 messenger RNA expression. Effects of each agonist were blocked by an AT2R antagonist (PD123319) and also by a MAS antagonist (A-779). Knockout of a single of these receptors made astrocytes unresponsive for both agonists. Our results suggest that MAS and the AT2R form heterodimers and that-at least in astrocytes-both receptors functionally depend on each other.

mTORC1 activity repression by late endosomal phosphatidylinositol 3,4-bisphosphate
Marat, A. L., Wallroth, A., Lo, W. T., Müller(*), R., Norata(*), G. D., Falasca(*), M., Schultz(*), C.; Haucke, V.
Science, 356:968-972

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Nutrient sensing by mechanistic target of rapamycin complex 1 (mTORC1) on lysosomes and late endosomes (LyLEs) regulates cell growth. Many factors stimulate mTORC1 activity, including the production of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] by class I phosphatidylinositol 3-kinases (PI3Ks) at the plasma membrane. We investigated mechanisms that repress mTORC1 under conditions of growth factor deprivation. We identified phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], synthesized by class II PI3K beta (PI3KC2beta) at LyLEs, as a negative regulator of mTORC1, whereas loss of PI3KC2beta hyperactivated mTORC1. Growth factor deprivation induced the association of PI3KC2beta with the Raptor subunit of mTORC1. Local PI(3,4)P2 synthesis triggered repression of mTORC1 activity through association of Raptor with inhibitory 14-3-3 proteins. These results unravel an unexpected function for local PI(3,4)P2 production in shutting off mTORC1.

Eighth International Chorea-Acanthocytosis Symposium: Summary of Workshop Discussion and Action Points
Pappas(*), S. S., Bonifacino(*), J., Danek(*), A., Dauer, W. T., De(*), M., De Franceschi(*), L., DiPaolo(*), G., Fuller(*), R., Haucke, V., Hermann(*), A., Kornmann(*), B., Landwehrmeyer(*), B., Levin(*), J., Neiman(*), A. M., Rudnicki(*), D. D., Sibon(*), O., Velayos-Baeza(*), A., Vonk(*), J. J., Walker(*), R. H., Weisman(*), L. S.; Albin(*), R. L.
Tremor and other hyperkinetic movements (New York, N.Y.), 7:428

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Chorea-Acanthocytosis (ChAc) is a rare hereditary neurological disorder characterized by abnormal movements, red blood cell pathology, and progressive neurodegeneration. Little is understood of the pathogenesis of ChAc and related disorders (collectively Neuroacanthocytosis). The Eighth International Chorea-Acanthocytosis Symposium was held in May 2016 in Ann Arbor, MI, USA, and focused on molecular mechanisms driving ChAc pathophysiology. Accompanying the meeting, members of the neuroacanthocytosis research community and other invited scientists met in a workshop to discuss the current understanding and next steps needed to better understand ChAc pathogenesis. These discussions identified several broad and critical needs for advancing ChAc research and patient care, and led to the definition of 18 specific action points related to functional and molecular studies, animal models, and clinical research. These action points, described below, represent tractable research goals to pursue for the next several years.

The complex co-translational processing of glycoprotein GP5 of type 1 porcine reproductive and respiratory syndrome virus
Thaa(*), B., Kaufer(*), S., Neumann(*), S. A., Peibst(*), B., Nauwynck(*), H., Krause, E.; Veit(*), M.
Virus Res, 240:112-120

Tags: Mass Spectrometry (Krause, E.)

Abstract: GP5 and M, the major membrane proteins of porcine reproductive and respiratory syndrome virus (PRRSV), are the driving force for virus budding and a target for antibodies. We studied co-translational processing of GP5 from an European PRRSV-1 strain. Using mass spectrometry, we show that in virus particles of a Lelystad variant, the signal peptide of GP5 was absent due to cleavage between glycine-34 and asparagine-35. This cleavage site removes an epitope for a neutralizing monoclonal antibody, but leaves intact another epitope recognized by neutralizing pig sera. Upon ectopic expression of this GP5 in cells, signal peptide cleavage was however inefficient. Complete cleavage occurred when cysteine-24 was changed to proline or an unused glycosylation site involving asparagine-35 was mutated. Insertion of proline at position 24 also caused carbohydrate attachment to asparagine-35. Glycosylation sites introduced downstream of residue 35 were used, but did not inhibit signal peptide processing. Co-expression of the M protein rescued this processing defect in GP5, suggesting a novel function of M towards GP5. We speculate that a complex interplay of the co-translational modifications of GP5 affect the N-terminal structure of the mature proteins and hence its antigenicity.

Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis
Walter(*), A. M., Müller(*), R., Tawfik(*), B., Wierda(*), K. D., Pinheiro(*), P. S., Nadler(*), A., McCarthy(*), A. W., Ziomkiewicz(*), I., Kruse(*), M., Reither(*), G., Rettig(*), J., Lehmann, M., Haucke, V., Hille(*), B., Schultz(*), C.; Sorensen(*), J. B.
Elife, 6

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging bypasses CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors.


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

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.

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

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.

Organophosphorus-mediated N-N bond formation: facile access to 3-amino-2H-indazoles
Bel Abed, H., Schöne, J., Christmann(*), M.; Nazare, M.
Org Biomol Chem, 14:8520-8528

Tags: Medicinal Chemistry (Nazare)

Abstract: A convenient and efficient strategy has been devised to access 3-amino-2H-indazole derivatives in two steps from readily available starting materials. The conversion of 2-nitrobenzonitriles to substituted benzamidines followed by an organophosphorus-mediated reductive cyclization and a subsequent N-N bond formation afforded 3-amino-2H-indazoles in good to excellent yields.

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

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
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