Max Delbruck Centrum fur Molekulare Medizin MDC
Max Delbruck Centrum fur Molekulare Medizin MDC
Lutter D.,Leibniz Institute for Molecular Pharmacology |
Lutter D.,Free University of Berlin |
Ullrich F.,Leibniz Institute for Molecular Pharmacology |
Lueck J.C.,Leibniz Institute for Molecular Pharmacology |
And 4 more authors.
Journal of Cell Science | Year: 2017
In response to swelling, mammalian cells release chloride and organic osmolytes through volume-regulated anion channels (VRACs). VRACs are heteromers of LRRC8A and other LRRC8 isoforms (LRRC8B to LRRC8E), which are co-expressed in HEK293 and most other cells. The spectrum of VRAC substrates and its dependence on particular LRRC8 isoforms remains largely unknown. We show that, besides the osmolytes taurine and myo-inositol, LRRC8 channels transport the neurotransmitters glutamate, aspartate and γ-aminobutyric acid (GABA) and the co-activator D-serine. HEK293 cells engineered to express defined subsets of LRRC8 isoforms were used to elucidate the subunit-dependence of transport. Whereas LRRC8D was crucial for the translocation of overall neutral compounds like myo-inositol, taurine and GABA, and sustained the transport of positively charged lysine, flux of negatively charged aspartate was equally well supported by LRRC8E. Disruption of LRRC8B or LRRC8C failed to decrease the transport rates of all investigated substrates, but their inclusion into LRRC8 heteromers influenced the substrate preference of VRAC. This suggested that individual VRACs can contain three or more different LRRC8 subunits, a conclusion confirmed by sequential coimmunoprecipitations. Our work suggests a composition-dependent role of VRACs in extracellular signal transduction. © 2017. Published by The Company of Biologists Ltd.
Morelli G.,Max Planck Institute For Infektionsbiologie |
Morelli G.,Max Planck Institute For Molekulare Genetik |
Song Y.,Beijing Institute of Microbiology and Epidemiology |
Song Y.,University College Cork |
And 28 more authors.
Nature Genetics | Year: 2010
Plague is a pandemic human invasive disease caused by the bacterial agent Yersinia pestis. We here report a comparison of 17 whole genomes of Y. pestis isolates from global sources. We also screened a global collection of 286 Y. pestis isolates for 933 SNPs using Sequenom MassArray SNP typing. We conducted phylogenetic analyses on this sequence variation dataset, assigned isolates to populations based on maximum parsimony and, from these results, made inferences regarding historical transmission routes. Our phylogenetic analysis suggests that Y. pestis evolved in or near China and spread through multiple radiations to Europe, South America, Africa and Southeast Asia, leading to country-specific lineages that can be traced by lineage-specific SNPs. All 626 current isolates from the United States reflect one radiation, and 82 isolates from Madagascar represent a second radiation. Subsequent local microevolution of Y. pestis is marked by sequential, geographically specific SNPs. © 2010 Nature America, Inc. All rights reserved.
Hubner C.A.,Friedrich - Schiller University of Jena |
Hubner C.A.,Universitatsklinikum Jena |
Schroeder B.C.,Max Delbruck Centrum fur Molekulare Medizin MDC |
Ehmke H.,Universitatsklinikum Hamburg Eppendorf
Pflugers Archiv European Journal of Physiology | Year: 2015
Recent studies suggest that primary changes in vascular resistance can cause sustained changes in arterial blood pressure. In this review, we summarize current knowledge about Cl− homeostasis in vascular smooth muscle cells. Within vascular smooth muscle cells, Cl− is accumulated above the electrochemical equilibrium, causing Cl− efflux, membrane depolarization, and increased contractile force when Cl− channels are opened. At least two different transport mechanisms contribute to raise [Cl−]i in vascular smooth muscle cells, anion exchange, and cation-chloride cotransport. Recent work suggests that TMEM16A-associated Ca2+-activated Cl− currents mediate Cl− efflux in vascular smooth muscle cells leading to vasoconstriction. Additional proteins associated with Cl− flux in vascular smooth muscle are bestrophins, which modulate vasomotion, the volume-activated LRRC8, and the cystic fibrosis transmembrane conductance regulator (CFTR). Cl− transporters and Cl− channels in vascular smooth muscle cells (VSMCs) significantly contribute to the physiological regulation of vascular tone and arterial blood pressure. © 2015, Springer-Verlag Berlin Heidelberg.
Willoughby D.,University of Cambridge |
Halls M.L.,University of Cambridge |
Everett K.L.,University of Cambridge |
Ciruela A.,University of Cambridge |
And 3 more authors.
Journal of Cell Science | Year: 2012
Adenylyl cyclase (AC) isoforms can participate in multimolecular signalling complexes incorporating A-kinase anchoring proteins (AKAPs). We recently identified a direct interaction between Ca2+-sensitive AC8 and plasma membrane-targeted AKAP79/150 (in cultured pancreatic insulin-secreting cells and hippocampal neurons), which attenuated the stimulation of AC8 by Ca2+ entry (Willoughby et al., 2010). Here, we reveal that AKAP79 recruits cAMP-dependent protein kinase (PKA) to mediate the regulatory effects of AKAP79 on AC8 activity. Modulation by PKA is a novel means of AC8 regulation, which may modulate or apply negative feedback to the stimulation of AC8 by Ca2+ entry. We show that the actions of PKA are not mediated indirectly via PKA-dependent activation of protein phosphatase 2A (PP2A) B56d subunits that associate with the N-terminus of AC8. By site-directed mutagenesis we identify Ser-112 as an essential residue for direct PKA phosphorylation of AC8 (Ser-112 lies within the N-terminus of AC8, close to the site of AKAP79 association). During a series of experimentally imposed Ca2+ oscillations, AKAP79-targeted PKA reduced the on-rate of cAMP production in wild-type but not non-phosphorylatable mutants of AC8, which suggests that the protein-protein interaction may provide a feedback mechanism to dampen the downstream consequences of AC8 activation evoked by bursts of Ca2+ activity. This finetuning of Ca2+-dependent cAMP dynamics by targeted PKA could be highly significant for cellular events that depend on the interplay of Ca2+ and cAMP, such as pulsatile hormone secretion and memory formation. © 2012. Published by The Company of Biologists Ltd.
Hoppmann C.,Leibniz Institute for Molecular Pharmacology |
Schmieder P.,Leibniz Institute for Molecular Pharmacology |
Domaing P.,Max Delbruck Centrum fur Molekulare Medizin MDC |
Vogelreiter G.,Leibniz Institute for Molecular Pharmacology |
And 5 more authors.
Angewandte Chemie - International Edition | Year: 2011
A twitch in time: Light-directed inhibition of the interaction between α-1-syntrophin and neuronal NO synthase (nNOS) in living skeletal muscle with a cell-permeable photoswitchable nNOS-derived peptide ligand results in photocontrol of muscle performance and nitric oxide signaling. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Voss F.K.,Leibniz Institute for Molecular Pharmacology |
Voss F.K.,Free University of Berlin |
Ullrich F.,Leibniz Institute for Molecular Pharmacology |
Ullrich F.,Free University of Berlin |
And 11 more authors.
Science | Year: 2014
Regulation of cell volume is critical for many cellular and organismal functions, yet the molecular identity of a key player, the volume-regulated anion channel VRAC, has remained unknown. A genome-wide small interfering RNA screen in mammalian cells identified LRRC8A as a VRAC component. LRRC8A formed heteromers with other LRRC8 multispan membrane proteins. Genomic disruption of LRRC8A ablated VRAC currents. Cells with disruption of all five LRRC8 genes required LRRC8A cotransfection with other LRRC8 isoforms to reconstitute VRAC currents. The isoform combination determined VRAC inactivation kinetics. Taurine flux and regulatory volume decrease also depended on LRRC8 proteins. Our work shows that VRAC defines a class of anion channels, suggests that VRAC is identical to the volume-sensitive organic osmolyte/anion channel VSOAC, and explains the heterogeneity of native VRAC currents.
Horner A.,Johannes Kepler University |
Goetz F.,Max Delbruck Centrum fur Molekulare Medizin MDC |
Tampe R.,Goethe University Frankfurt |
Klussmann E.,Max Delbruck Centrum fur Molekulare Medizin MDC |
Pohl P.,Johannes Kepler University
Journal of Biological Chemistry | Year: 2012
A-kinase anchoring proteins (AKAPs) are a family of scaffolding proteins that target PKA and other signaling molecules to cellular compartments and thereby spatiotemporally define cellular signaling events. The AKAP18 family comprises AKAP18α, AKAP18β, AKAP18γ, and AKAP18δ. The δ isoform targets PKA and phosphodiesterase PDE4D to AQP2 (aquaporin-2)-bearing vesicles to orchestrate the acute regulation of body water balance. Therefore, AKAP18δ must adopt a membrane localization that seems at odds with (i) its lack of palmitoylation or myristoylation sites that tailor its isoforms AKAP18α and AKAP18β to membrane compartments and (ii) the high sequence identity to the preferentially cytoplasmic AKAP18γ. Here, we show that the electrostatic attraction of the positively charged amino acids of AKAP18δ to negatively charged lipids explains its membrane targeting. As revealed by fluorescence correlation spectroscopy, the binding constant of purified AKAP18δ fragments to large unilamellar vesicles correlates (i) with the fraction of net negatively charged lipids in the bilayer and (ii) with the total amount of basic residues in the protein. Although distantly located on the sequence, these positively charged residues concentrate in the tertiary structure and form a clear binding surface. Thus, specific recruitment of the AKAP18δ-based signaling module to membranes such as those of AQP2-bearing vesicles must be achieved by additional mechanisms, most likely compartment-specific protein-protein interactions. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
Maass P.G.,Max Delbruck Centrum fur Molekulare Medizin MDC
Medizinische Genetik | Year: 2014
Long non-coding RNAs (lncRNAs) expand our knowledge of transcriptional or posttranscriptional gene regulation. In interaction with the nuclear architecture, lncRNAs are involved in fundamental biological mechanisms, such as imprinting, histone code regulation, gene activation, gene repression, lineage determinations, and cell proliferation. Associations with apparent phenotypes have been attributed to lncRNAs. The involvement of lncRNA in gene regulation and disease underscores the importance of lncRNA-mediated regulatory networks. Manipulating lncRNAs is a conceivable therapeutic strategy because of their tissue-specific expression and their selective target genes. © 2014 Springer-Verlag.
Michalik K.M.,Goethe University Frankfurt |
You X.,Max Delbruck Centrum fur Molekulare Medizin MDC |
Manavski Y.,Goethe University Frankfurt |
Doddaballapur A.,Goethe University Frankfurt |
And 10 more authors.
Circulation Research | Year: 2014
Rationale: The human genome harbors a large number of sequences encoding for RNAs that are not translated but control cellular functions by distinct mechanisms. The expression and function of the longer transcripts namely the long noncoding RNAs in the vasculature are largely unknown. OBJECTIVE:: Here, we characterized the expression of long noncoding RNAs in human endothelial cells and elucidated the function of the highly expressed metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). METHODS AND RESULTS:: Endothelial cells of different origin express relative high levels of the conserved long noncoding RNAs MALAT1, taurine upregulated gene 1 (TUG1), maternally expressed 3 (MEG3), linc00657, and linc00493. MALAT1 was significantly increased by hypoxia and controls a phenotypic switch in endothelial cells. Silencing of MALAT1 by small interfering RNAs or GapmeRs induced a promigratory response and increased basal sprouting and migration, whereas proliferation of endothelial cells was inhibited. When angiogenesis was further stimulated by vascular endothelial growth factor, MALAT1 small interfering RNAs induced discontinuous sprouts indicative of defective proliferation of stalk cells. In vivo studies confirmed that genetic ablation of MALAT1 inhibited proliferation of endothelial cells and reduced neonatal retina vascularization. Pharmacological inhibition of MALAT1 by GapmeRs reduced blood flow recovery and capillary density after hindlimb ischemia. Gene expression profiling followed by confirmatory quantitative reverse transcriptase-polymerase chain reaction demonstrated that silencing of MALAT1 impaired the expression of various cell cycle regulators. CONCLUSIONS:: Silencing of MALAT1 tips the balance from a proliferative to a migratory endothelial cell phenotype in vitro, and its genetic deletion or pharmacological inhibition reduces vascular growth in vivo. © 2014 American Heart Association, Inc.
Max Delbruck Centrum Fur Molekulare Medizin Mdc | Date: 2013-11-25
The present invention relates to methods for isolating human forkhead box P3 (Foxp3+) CD4+ regulatory T cells (herein referred to a Foxp3+ Treg cells) from a sample containing (i) peripheral blood mononuclear cells (PBMCs), (ii) a lymphocyte containing fluid, or (iii) a lymphocyte containing tissue, a kit for isolating human Foxp3+ Treg cells, and the use of anti-CD49d antibody for the isolation of human Foxp3+ Treg cells.