FMP Publications

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

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References

2017

Post-translational cleavage of Hv1 in human sperm tunes pH- and voltage-dependent gating
Berger(*), T. K., Fusshöller(*), D. M., Goodwin(*), N., Bönigk(*), W., Müller(*), A., Dokani Khesroshahi(*), N., Brenker(*), C., Wachten(*), D., Krause, E., Kaupp(*), U. B.; Strünker(*), T.
J Physiol, 595:1533-1546
(2017)

Tags: Mass Spectrometry (Krause, E.)

Abstract: KEY POINTS: In human sperm, proton flux across the membrane is controlled by the voltage-gated proton channel Hv1. We show that sperm harbour both Hv1 and an N-terminally cleaved isoform termed Hv1Sper. The pH-control of Hv1Sper and Hv1 is distinctively different. Hv1Sper and Hv1 can form heterodimers that combine features of both constituents. Cleavage and heterodimerization of Hv1 might represent an adaptation to the specific requirements of pH control in sperm. ABSTRACT: In human sperm, the voltage-gated proton channel Hv1 controls the flux of protons across the flagellar membrane. Here, we show that sperm harbour Hv1 and a shorter isoform, termed Hv1Sper. Hv1Sper is generated from Hv1 by removal of 68 amino acids from the N-terminus by post-translational proteolytic cleavage. The pH-dependent gating of the channel isoforms is distinctly different. In both Hv1 and Hv1Sper, the conductance-voltage relationship is determined by the pH difference across the membrane (pH). However, simultaneous changes in intracellular and extracellular pH that leave DeltapH constant strongly shift the activation curve of Hv1Sper but not that of Hv1, demonstrating that cleavage of the N-terminus tunes pH sensing in Hv1. Moreover, we show that Hv1 and Hv1Sper assemble as heterodimers that combine features of both constituents. We suggest that cleavage and heterodimerization of Hv1 represents an adaptation to the specific requirements of pH control in sperm.

Claudin peptidomimetics modulate tissue barriers for enhanced drug delivery
Dithmer, S., Staat, C., Müller, C., Ku(*), M. C., Pohlmann(*), A., Niendorf(*), T., Gehne, N., Fallier-Becker(*), P., Kittel(*), A., Walter(*), F. R., Veszelka(*), S., Deli(*), M. A., Blasig, R., Haseloff, R. F., Blasig, I. E.; Winkler, L.
Annals of the New York Academy of Sciences, 1397:169-184
(2017)

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

Abstract: The blood-brain barrier (BBB) formed by the microvascular endothelium limits cerebral drug delivery. The paraendothelial cleft is sealed by tight junctions (TJs) with a major contribution from claudin-5, which we selected as a target to modulate BBB permeability. For this purpose, drug-enhancer peptides were designed based on the first extracellular loop (ECL) of claudin-5 to allow transient BBB permeabilization. Peptidomimetics (C5C2 and derivatives, nanomolar affinity to claudin-5) size-selectively (</=40 kDa) and reversibly (12-48 h) increased the permeability of brain endothelial and claudin-5-transfected epithelial cell monolayers. Upon peptide uptake, the number of TJ strand particles diminished, claudin-5 was downregulated and redistributed from cell-cell contacts to the cytosol, and the cell shape was altered. Cellular permeability of doxorubicin (cytostatic drug, 580 Da) was enhanced after peptide administration. Mouse studies (3.5 mumol/kg i.v.) confirmed that, for both C5C2 and a d-amino acid derivative, brain uptake of Gd-diethylene-triamine penta-acetic acid (547 Da) was enhanced within 4 h of treatment. On the basis of our functional data, circular dichroism measurements, molecular modeling, and docking experiments, we suggest an association model between beta-sheets flanked by alpha-helices, formed by claudin-5 ECLs, and the peptides. In conclusion, we identified claudin-5 peptidomimetics that improve drug delivery through endothelial and epithelial barriers expressing claudin-5.

A Chemical Disruptor of the ClpX Chaperone Complex Attenuates Multiresistant Staphylococcus aureus Virulence
Fetzer(*), C., Korotkov(*), V. S., Thanert(*), R., Lee(*), K. M., Neuenschwander, M., von Kries, J. P., Medina(*), E.; Sieber(*), S. A.
Angew Chem Int Ed Engl,
(2017)

Tags: Screening Unit (von Kries)

Abstract: The Staphylococcus aureus ClpXP protease is an important regulator of cell homeostasis and virulence. Here we utilize a high-throughput screen against the ClpXP complex and identify a specific inhibitor of the ClpX chaperone that disrupts its oligomeric state. Synthesis of 34 derivatives revealed that the molecular scaffold is restrictive for diversification with only minor changes tolerated. Subsequent analysis of the most active compound revealed strong attenuation of S. aureus toxin production which was quantified via a customized MS-based assay platform. Transcriptome and whole proteome studies further confirmed the global reduction of virulence and unraveled characteristic signatures of protein expression in compound treated cells. Although these partially matched the pattern of ClpX knockout cells, further depletion of toxins was observed leading to the intriguing perspective that additional virulence pathways may be directly or indirectly addressed by the small molecule.

Complete NMR assignment and conformational analysis of 17-alpha-ethinylestradiol by using RDCs obtained in grafted graphene oxide
Franca(*), J. A., Navarro-Vazquez(*), A., Lei(*), X., Sun, H., Griesinger(*), C.; Hallwass(*), F.
Magn Reson Chem, 55:297-303
(2017)

Tags: Computational Chemistry and Protein Design (Kühne)

Abstract: The 1 H and 13 C NMR spectra of 17-alpha-ethinylestradiol (EE2), a well-known contraceptive, including diastereotopic methylene groups, were fully assigned with the help of residual dipolar couplings (RDC) measured in the recently developed grafted graphene oxide orienting medium. RDC analysis, which included all 1 DCH couplings and the long-range 2 DCH1 H-C identical with13 C coupling, also pointed to the presence of a minor conformation arising from pseudo-rotation of the steroid B ring. Saturation-transfer difference (STD) measurements revealed that the most likely interaction between EE2 and orienting medium occurred on the C and D ring. Copyright (c) 2016 John Wiley & Sons, Ltd.

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells
Herce(*), H. D., Schumacher, D., Schneider, A. F. L., Ludwig(*), A. K., Mann, F. A., Fillies(*), M., Kasper, M. A., Reinke, S., Krause, E., Leonhardt(*), H., Cardoso(*), M. C.; Hackenberger, C. P. R.
Nature chemistry, 9:762-771
(2017)

Tags: Chemical Biology II (Hackenberger), Mass Spectrometry (Krause, E)

Abstract: Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

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
(2017)

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.

Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders
Lorenz(*), C., Lesimple(*), P., Bukowiecki(*), R., Zink(*), A., Inak(*), G., Mlody(*), B., Singh(*), M., Semtner(*), M., Mah(*), N., Aure(*), K., Leong(*), M., Zabiegalov(*), O., Lyras(*), E. M., Pfiffer(*), V., Fauler(*), B., Eichhorst, J., Wiesner, B., Huebner(*), N., Priller(*), J., Mielke(*), T., Meierhofer(*), D., Izsvak(*), Z., Meier(*), J. C., Bouillaud(*), F., Adjaye(*), J., Schuelke(*), M., Wanker(*), E. E., Lombes(*), A.; Prigione(*), A.
Cell stem cell,
(2017)

Tags: Cellular Imaging (Wiesner)

Abstract: Mitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Modeling of these defects has been difficult because of the challenges associated with engineering mtDNA. We show here that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) retain the parental mtDNA profile and exhibit a metabolic switch toward oxidative phosphorylation. NPCs derived in this way from patients carrying a deleterious homoplasmic mutation in the mitochondrial gene MT-ATP6 (m.9185T>C) showed defective ATP production and abnormally high mitochondrial membrane potential (MMP), plus altered calcium homeostasis, which represents a potential cause of neural impairment. High-content screening of FDA-approved drugs using the MMP phenotype highlighted avanafil, which we found was able to partially rescue the calcium defect in patient NPCs and differentiated neurons. Overall, our results show that iPSC-derived NPCs provide an effective model for drug screening to target mtDNA disorders that affect the nervous system.

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
(2017)

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.

Regulation of body weight and energy homeostasis by neuronal cell adhesion molecule 1
Rathjen(*), T., Yan(*), X., Kononenko, N. L., Ku(*), M. C., Song(*), K., Ferrarese(*), L., Tarallo(*), V., Puchkov, D., Kochlamazashvili, G., Brachs(*), S., Varela(*), L., Szigeti-Buck(*), K., Yi(*), C. X., Schriever(*), S. C., Tattikota(*), S. G., Carlo(*), A. S., Moroni(*), M., Siemens(*), J., Heuser(*), A., van der Weyden(*), L., Birkenfeld(*), A. L., Niendorf(*), T., Poulet(*), J. F. A., Horvath(*), T. L., Tschop(*), M. H., Heinig(*), M., Trajkovski(*), M., Haucke, V.; Poy(*), M. N.
Nat Neurosci,
(2017)

Tags: Molecular Pharmacology and Cell Biology (Haucke), Cellular Imaging (Wiesner, Puchkov)

Abstract: Susceptibility to obesity is linked to genes regulating neurotransmission, pancreatic beta-cell function and energy homeostasis. Genome-wide association studies have identified associations between body mass index and two loci near cell adhesion molecule 1 (CADM1) and cell adhesion molecule 2 (CADM2), which encode membrane proteins that mediate synaptic assembly. We found that these respective risk variants associate with increased CADM1 and CADM2 expression in the hypothalamus of human subjects. Expression of both genes was elevated in obese mice, and induction of Cadm1 in excitatory neurons facilitated weight gain while exacerbating energy expenditure. Loss of Cadm1 protected mice from obesity, and tract-tracing analysis revealed Cadm1-positive innervation of POMC neurons via afferent projections originating from beyond the arcuate nucleus. Reducing Cadm1 expression in the hypothalamus and hippocampus promoted a negative energy balance and weight loss. These data identify essential roles for Cadm1-mediated neuronal input in weight regulation and provide insight into the central pathways contributing to human obesity.

Small-molecule inhibition of STOML3 oligomerization reverses pathological mechanical hypersensitivity
Wetzel(*), C., Pifferi(*), S., Picci(*), C., Gök(*), C., Hoffmann(*), D., Bali(*), K. K., Lampe, A., Lapatsina(*), L., Fleischer(*), R., Smith(*), E. S., Begay(*), V., Moroni(*), M., Estebanez(*), L., Kühnemund(*), J., Walcher(*), J., Specker, E., Neuenschwander, M., von Kries, J. P., Haucke, V., Kuner(*), R., Poulet(*), J. F., Schmoranzer(*), J., Poole(*), K.; Lewin(*), G. R.
Nat Neurosci, 20:209-218
(2017)

Tags: Molecular Pharmacology and Cell Biology (Haucke), Screening Unit (von Kries)

Abstract: The skin is equipped with specialized mechanoreceptors that allow the perception of the slightest brush. Indeed, some mechanoreceptors can detect even nanometer-scale movements. Movement is transformed into electrical signals via the gating of mechanically activated ion channels at sensory endings in the skin. The sensitivity of Piezo mechanically gated ion channels is controlled by stomatin-like protein-3 (STOML3), which is required for normal mechanoreceptor function. Here we identify small-molecule inhibitors of STOML3 oligomerization that reversibly reduce the sensitivity of mechanically gated currents in sensory neurons and silence mechanoreceptors in vivo. STOML3 inhibitors in the skin also reversibly attenuate fine touch perception in normal mice. Under pathophysiological conditions following nerve injury or diabetic neuropathy, the slightest touch can produce pain, and here STOML3 inhibitors can reverse mechanical hypersensitivity. Thus, small molecules applied locally to the skin can be used to modulate touch and may represent peripherally available drugs to treat tactile-driven pain following neuropathy.

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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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