FMP Publications

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

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Improved intracellular delivery of peptide- and lipid-nanoplexes by natural glycosides
Weng(*), A., Manunta(*), M. D., Thakur(*), M., Gilabert-Oriol(*), R., Tagalakis(), A. D., Eddaoudi(*), A., Munye(*), M. M., Vink(*), C. A., Wiesner, B., Eichhorst, J., Melzig(*), M. F.; Hart(*), S. L.
J Control Release, 206:75-90

Tags: Cellular Imaging (Wiesner)

Abstract: Targeted nanocarriers undergo endocytosis upon binding to their membrane receptors and are transported into cellular compartments such as late endosomes and lysosomes. In gene delivery the genetic material has to escape from the cellular compartments into the cytosol. The process of endosomal escape is one of the most critical steps for successful gene delivery. For this reason synthetic lipids with fusogenic properties such as 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) are integrated into the nanocarriers. In this study we show that a natural, plant derived glycoside (SO1861) from Saponaria officinalis L. greatly improves the efficacy of lipid based as well as non-lipid based targeted nanoplexes consisting of a targeted K16 peptide with a nucleic acid binding domain and plasmid-DNA, minicircle-DNA or small interfering RNA (siRNA). By confocal live cell imaging and single cell analyses, we demonstrate that SO1861 augments the escape of the genetic cargo out of the intracellular compartments into the cytosol. Co-localisation experiments with fluorescence labelled dextran and transferrin indicate that SO1861 induces the release of the genetic cargo out of endosomes and lysosomes. However, the transduction efficacy of a lentivirus based gene delivery system was not augmented. In order to design receptor-targeted nanoplexes (LPD) with improved functional properties, SO1861 was integrated into the lipid matrix of the LPD. The SO1861 sensitized LPD (LPDS) were characterized by dynamic light scattering and transmission electron microscopy. Compared to their LPD counterparts the LPDS-nanoplexes showed a greatly improved gene delivery. As shown by differential scanning calorimetry SO1861 can be easily integrated into the lipid bilayer of glycerophospholipid model membranes. This underlines the great potential of SO1861 as a new transfection multiplier for non-viral gene delivery systems.

Sensitivity and resolution of proton detected spectra of a deuterated protein at 40 and 60 kHz magic-angle-spinning
Nieuwkoop, A. J., Franks, W. T., Rehbein, K., Diehl, A., Akbey, Ü., Engelke(*), F., Emsley(*), L., Pintacuda(*), G.; Oschkinat, H.
J Biomol NMR, 61:161-171

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: The use of small rotors capable of very fast magic-angle spinning (MAS) in conjunction with proton dilution by perdeuteration and partial reprotonation at exchangeable sites has enabled the acquisition of resolved, proton detected, solid-state NMR spectra on samples of biological macromolecules. The ability to detect the high-gamma protons, instead of carbons or nitrogens, increases sensitivity. In order to achieve sufficient resolution of the amide proton signals, rotors must be spun at the maximum rate possible given their size and the proton back-exchange percentage tuned. Here we investigate the optimal proton back-exchange ratio for triply labeled SH3 at 40 kHz MAS. We find that spectra acquired on 60 % back-exchanged samples in 1.9 mm rotors have similar resolution at 40 kHz MAS as spectra of 100 % back-exchanged samples in 1.3 mm rotors spinning at 60 kHz MAS, and for (H)NH 2D and (H)CNH 3D spectra, show 10-20 % higher sensitivity. For 100 % back-exchanged samples, the sensitivity in 1.9 mm rotors is superior by a factor of 1.9 in (H)NH and 1.8 in (H)CNH spectra but at lower resolution. For (H)C(C)NH experiments with a carbon-carbon mixing period, this sensitivity gain is lost due to shorter relaxation times and less efficient transfer steps. We present a detailed study on the sensitivity of these types of experiments for both types of rotors, which should enable experimentalists to make an informed decision about which type of rotor is best for specific applications.

Vesicle uncoating regulated by SH3-SH3 domain-mediated complex formation between endophilin and intersectin at synapses
Pechstein, A., Gerth(*), F., Milosevic(*), I., Jäpel, M., Eichhorn-Grünig, M., Vorontsova(*), O., Bacetic, J., Maritzen, T., Shupliakov(*), O., Freund(*), C.; Haucke, V.
Embo Rep, 16:232-239

Tags: Molecular Pharmacology and Cell Biology (Haucke), Membrane Traffic and Cell Motility (Maritzen)

Abstract: Neurotransmission involves the exo-endocytic cycling of synaptic vesicle (SV) membranes. Endocytic membrane retrieval and clathrin-mediated SV reformation require curvature-sensing and membrane-bending BAR domain proteins such as endophilin A. While their ability to sense and stabilize curved membranes facilitates membrane recruitment of BAR domain proteins, the precise mechanisms by which they are targeted to specific sites of SV recycling has remained unclear. Here, we demonstrate that the multi-domain scaffold intersectin 1 directly associates with endophilin A to facilitate vesicle uncoating at synapses. Knockout mice deficient in intersectin 1 accumulate clathrin-coated vesicles at synapses, a phenotype akin to loss of endophilin function. Intersectin 1/endophilin A1 complex formation is mediated by direct binding of the SH3B domain of intersectin to a non-canonical site on the SH3 domain of endophilin A1. Consistent with this, intersectin-binding defective mutant endophilin A1 fails to rescue clathrin accumulation at neuronal synapses derived from endophilin A1-3 triple knockout (TKO) mice. Our data support a model in which intersectin aids endophilin A recruitment to sites of clathrin-mediated SV recycling, thereby facilitating vesicle uncoating.

Phosphoinositides in endocytosis
Posor, Y., Eichhorn-Grünig, M.; Haucke, V.
Biochim Biophys Acta, 1851:794-804

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: The internalization and subsequent endosomal trafficking of proteins and membrane along the endocytic pathway is a fundamental cellular process. Over the last two decades, this pathway has emerged to be subject to extensive regulation by phosphoinositides (PIs), phosphorylated derivatives of the minor membrane phospholipid phosphatidylinositol. Clathrin-mediated endocytosis (CME) is the endocytic mechanism characterized in most detail. It now represents a prime example of a process spatiotemporally organized by the interplay of PI metabolizing enzymes. The most abundant PI, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)], serves as a denominator of plasma membrane identity and together with cargo proteins is instrumental for the initiation of clathrin-coated pit (CCP) formation. During later stages of the process, the generation of phosphatidylinositol-3,4-bisphosphate [PI(3,4)P(2)] and the dephosphorylation of PI(4,5)P(2)regulate CCP maturation and vesicle uncoating. Here we provide an overview of the mechanisms by which PIs are made and consumed to regulate CME and other endocytic pathways and how conversion of PIs en route to endosomes may be accomplished. Mutations in PI converting enzymes are linked to multiple diseases ranging from mental retardation and neurodegeneration, to inherited muscle and kidney disorders suggesting that the tight control of PI levels along the endocytic pathway plays a critical role in cell physiology. This article is part of a Special Issue entitled Phosphoinositides.

Lipids, GTPases, and their regulators in membrane dynamics: an intracellular menage a trois
Echard(*), A.; Haucke, V.
Mol Biol Cell, 26:1012-1013

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Postsynaptic D2 dopamine receptor supersensitivity in the striatum of mice lacking TAAR1
Espinoza(*), S., Ghisi, V., Emanuele(*), M., Leo(*), D., Sukhanov(*), I., Sotnikova(*), T. D., Chieregatti(*), E.; Gainetdinov(*), R. R.
Neuropharmacology, 93:308-313

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: Trace Amine-Associated Receptor 1 (TAAR1) is a G protein-coupled receptor (GPCR) known to modulate dopaminergic system through several mechanisms. Mice lacking this receptor show a higher sensitivity to dopaminergic stimuli, such as amphetamine; however, it is not clear whether D1 or D2 dopamine receptors and which associated intracellular signaling events are involved in this modulation. In the striatum of TAAR1 knock out (TAAR1-KO mice) we found that D2, but not D1, dopamine receptors were over-expressed, both in terms of mRNA and protein levels. Moreover, the D2 dopamine receptor-related G protein-independent AKT/GSK3 signaling pathway was selectively activated, as indicated by the decrease of phosphorylation of AKT and GSK3beta. The decrease in phospho-AKT levels, suggesting an increase in D2 dopamine receptor activity in basal conditions, was associated with an increase of AKT/PP2A complex, as revealed by co-immunoprecipitation experiments. Finally, we found that the locomotor activation induced by the D2 dopamine receptor agonist quinpirole, but not by the full D1 dopamine receptor agonist SKF-82958, was increased in TAAR1-KO mice. These data demonstrate pronounced supersensitivity of postsynaptic D2 dopamine receptors in the striatum of TAAR1-KO mice and indicate that a close interaction of TAAR1 and D2 dopamine receptors at the level of postsynaptic structures has important functional consequences.

Selective inhibitors of the protein tyrosine phosphatase SHP2 block cellular motility and growth of cancer cells in vitro and in vivo
Grosskopf, S., Eckert, C., Arkona(*), C., Radetzki, S., Böhm(*), K., Heinemann(*), U., Wolber(*), G., von Kries, J. P., Birchmeier(*), W.; Rademann(*), J.
Chemmedchem, 10:815-826

Tags: Medicinal Chemistry (Rademann), Screening Unit (von Kries)

Abstract: Selective inhibitors of the protein tyrosine phosphatase SHP2 (src homology region 2 domain phosphatase; PTPN11), an enzyme that is deregulated in numerous human tumors, were generated through a combination of chemical synthesis and structure-based rational design. Seventy pyridazolon-4-ylidenehydrazinyl benzenesulfonates were prepared and evaluated in enzyme assays. The binding modes of active inhibitors were simulated in silico using a newly generated crystal structure of SHP2. The most powerful compound, GS-493 (4-(2Z)-2-[1,3-bis(4-nitrophenyl)-5-oxo-1,5-dihydro-4H-pyrazol-4-yliden]hydrazin obenzenesulfonic acid; 25) inhibited SHP2 with an IC50 value of 71+/-15 nM in the enzyme assay and was 29- and 45-fold more active toward SHP2 than against related SHP1 and PTP1B. In cell culture experiments compound 25 was found to block hepatocyte growth factor (HGF)-stimulated epithelial-mesenchymal transition of human pancreatic adenocarcinoma (HPAF) cells, as indicated by a decrease in the minimum neighbor distances of cells. Moreover, 25 inhibited cell colony formation in the non-small-cell lung cancer cell line LXFA 526L in soft agar. Finally, 25 was observed to inhibit tumor growth in a murine xenograft model. Therefore, the novel specific compound 25 strengthens the hypothesis that SHP2 is a relevant protein target for the inhibition of mobility and invasiveness of cancer cells.

Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs
Planells-Cases, R., Lutter, D., Guyader(*), C., Gerhards(*), N. M., Ullrich, F., Elger, D. A., Kucukosmanoglu(*), A., Xu(*), G., Voss, F. K., Reincke, S. M., Stauber, T., Blomen(*), V. A., Vis(*), D. J., Wessels(*), L. F., Brummelkamp(*), T. R., Borst(*), P., Rottenberg(*), S.; Jentsch, T. J.
EMBO J, 34:2993-3008

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Although platinum-based drugs are widely used chemotherapeutics for cancer treatment, the determinants of tumor cell responsiveness remain poorly understood. We show that the loss of subunits LRRC8A and LRRC8D of the heteromeric LRRC8 volume-regulated anion channels (VRACs) increased resistance to clinically relevant cisplatin/carboplatin concentrations. Under isotonic conditions, about 50% of cisplatin uptake depended on LRRC8A and LRRC8D, but neither on LRRC8C nor on LRRC8E. Cell swelling strongly enhanced LRRC8-dependent cisplatin uptake, bolstering the notion that cisplatin enters cells through VRAC. LRRC8A disruption also suppressed drug-induced apoptosis independently from drug uptake, possibly by impairing VRAC-dependent apoptotic cell volume decrease. Hence, by mediating cisplatin uptake and facilitating apoptosis, VRAC plays a dual role in the cellular drug response. Incorporation of the LRRC8D subunit into VRAC substantially increased its permeability for cisplatin and the cellular osmolyte taurine, indicating that LRRC8 proteins form the channel pore. Our work suggests that LRRC8D-containing VRACs are crucial for cell volume regulation by an important organic osmolyte and may influence cisplatin/carboplatin responsiveness of tumors.

Directed structural modification of Clostridium perfringens enterotoxin to enhance binding to claudin-5
Protze, J., Eichner, M., Piontek, A., Dinter, S., Rossa, J., Blecharz(*), K. G., Vajkoczy(*), P., Piontek(*), J.; Krause, G.
Cellular and molecular life sciences : CMLS, 72:1417-1432

Tags: Structural Bioinformatics and Protein Design (Krause, G.)

Abstract: Clostridium perfringens enterotoxin (CPE) binds to distinct claudins (Clds), which regulate paracellular barrier functions in endo- and epithelia. The C-terminal domain (cCPE) has the potential for selective claudin modulation, since it only binds to a subset of claudins, e.g., Cld3 and Cld4 (cCPE receptors). Cld5 (non-CPE receptor) is a main constituent in tight junctions (TJ) of the blood-brain barrier. We aimed to reveal claudin recognition mechanisms of cCPE and to create a basis for a Cld5-binder. By utilizing structure-based interaction models, mutagenesis and assays of cCPE-binding to the TJ-free cell line HEK293, transfected with human Cld1 and murine Cld5, we showed how cCPE-binding to Cld1 and Cld5 is prevented by two residues in extracellular loop 2 of Cld1 (Asn(150) and Thr(153)) and Cld5 (Asp(149) and Thr(151)). Binding to Cld5 is especially attenuated by the lack of a bulky hydrophobic residue like leucine at position 151. By downsizing the binding pocket and compensating for the lack of this leucine residue, we created a novel cCPE-variant; cCPEY306W/S313H binds Cld5 with nanomolar affinity (K d 33 +/- 10 nM). Finally, the effective binding to endogenously Cld5-expressing blood-brain barrier model cells (murine microvascular endothelial cEND cell line) suggests cCPEY306W/S313H as basis for Cld5-specific modulation to improve paracellular drug delivery, or to target claudin overexpressing tumors.

Mode of action of claudin peptidomimetics in the transient opening of cellular tight junction barriers
Staat, C., Coisne(*), C., Dabrowski, S., Stamatovic(*), S. M., Andjelkovic(*), A. V., Wolburg(*), H., Engelhardt(*), B.; Blasig, I. E.
Biomaterials, 54:9-20

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

Abstract: In epithelial/endothelial barriers, claudins form tight junctions, seal the paracellular cleft, and limit the uptake of solutes and drugs. The peptidomimetic C1C2 from the C-terminal half of claudin-1's first extracellular loop increases drug delivery through epithelial claudin-1 barriers. However, its molecular and structural mode of action remains unknown. In the present study, >100 muM C1C2 caused paracellular opening of various barriers with different claudin compositions, ranging from epithelial to endothelial cells, preferentially modulating claudin-1 and claudin-5. After 6 h incubation, C1C2 reversibly increased the permeability to molecules of different sizes; this was accompanied by redistribution of claudins and occludin from junctions to cytosol. Internalization of C1C2 in epithelial cells depended on claudin-1 expression and clathrin pathway, whereby most C1C2 was retained in recyclosomes >2 h. In freeze-fracture electron microscopy, C1C2 changed claudin-1 tight junction strands to a more parallel arrangement and claudin-5 strands from E-face to P-face association - drastic and novel effects. In conclusion, C1C2 is largely recycled in the presence of a claudin, which explains the delayed onset of barrier and junction loss, the high peptide concentration required and the long-lasting effect. Epithelial/endothelial barriers are specifically modulated via claudin-1/claudin-5, which can be targeted to improve drug delivery.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)

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