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Freedberg D.I.,U.S. Food and Drug Administration | Selenko P.,Leibniz Institute for Molecular Pharmacology
Annual Review of Biophysics | Year: 2014

Ever since scientists realized that cells are the basic building blocks of all life, they have been developing tools to look inside them to reveal the architectures and mechanisms that define their biological functions. Whereas "looking into cells" is typically said in reference to optical microscopy, high-resolution in-cell and on-cell nuclear magnetic resonance (NMR) spectroscopy is a powerful method that offers exciting new possibilities for structural and functional studies in and on live cells. In contrast to conventional imaging techniques, in- and on-cell NMR methods do not provide spatial information on cellular biomolecules. Instead, they enable atomic-resolution insights into the native cell states of proteins, nucleic acids, glycans, and lipids. Here we review recent advances and developments in both fields and discuss emerging concepts that have been delineated with these methods. Copyright © 2014 by Annual Reviews. All rights reserved.


Jentsch T.J.,Leibniz Institute for Molecular Pharmacology
Nature Reviews Molecular Cell Biology | Year: 2016

Cells need to regulate their volume to counteract osmotic swelling or shrinkage, as well as during cell division, growth, migration and cell death. Mammalian cells adjust their volume by transporting potassium, sodium, chloride and small organic osmolytes using plasma membrane channels and transporters. This generates osmotic gradients, which drive water in and out of cells. Key players in this process are volume-regulated anion channels (VRACs), the composition of which has recently been identified and shown to encompass LRRC8 heteromers. VRACs also transport metabolites and drugs and function in extracellular signal transduction, apoptosis and anticancer drug resistance. © 2016 Macmillan Publishers Limited. All rights reserved.


Schumacher D.,Leibniz Institute for Molecular Pharmacology | Hackenberger C.P.,Leibniz Institute for Molecular Pharmacology
Current opinion in chemical biology | Year: 2014

The quest to enlarge the molecular space of functional biomolecules has led to the discovery of selective, mild and high-yielding chemical reactions for the modification of peptides and proteins. These conjugation methods have recently become even more advanced with the advent of modern biochemical techniques such as unnatural protein expression or enzymatic reactions that allow the site-specific modification of proteins. Within this overview, we will highlight recent examples that describe the site-specific functionalization of proteins. These examples go beyond the straightforward attachment of a given functional moiety to the protein backbone by employing either an innovative linker-design or by novel conjugation chemistry, where the modification reaction itself is responsible for the (altered) functional behaviour of the biomolecule. The examples covered herein include 'turn-on' probes for cellular imaging with low levels of background fluorescence, branched or cleavable polymer-protein conjugates of high stability within a cellular environment, the installation of natural occurring posttranslational modifications to help understand their role in complex cellular environments and finally the engineering of novel antibody drug conjugates to facilitate target specific drug release. Copyright © 2014 Elsevier Ltd. All rights reserved.


Jentsch T.J.,Leibniz Institute for Molecular Pharmacology
Journal of Physiology | Year: 2015

After providing a personal description of the convoluted path leading 25 years ago to the molecular identification of the Torpedo Cl- channel ClC-0 and the discovery of the CLC gene family, I succinctly describe the general structural and functional features of these ion transporters before giving a short overview of mammalian CLCs. These can be categorized into plasma membrane Cl- channels and vesicular Cl-/H+-exchangers. They are involved in the regulation of membrane excitability, transepithelial transport, extracellular ion homeostasis, endocytosis and lysosomal function. Diseases caused by CLC dysfunction include myotonia, neurodegeneration, deafness, blindness, leukodystrophy, male infertility, renal salt loss, kidney stones and osteopetrosis, revealing a surprisingly broad spectrum of biological roles for chloride transport that was unsuspected when I set out to clone the first voltage-gated chloride channel. © 2015 The Physiological Society.


Ito Y.,Tokyo Metroplitan University | Selenko P.,Leibniz Institute for Molecular Pharmacology
Current Opinion in Structural Biology | Year: 2010

While we appreciate the complexity of the intracellular environment as a general property of every living organism, we collectively lack the appropriate tools to analyze protein structures in a cellular context. In-cell NMR spectroscopy represents a novel biophysical tool to investigate the conformational and functional characteristics of biomolecules at the atomic level inside live cells. Here, we review recent in-cell NMR developments and provide an outlook towards future applications in prokaryotic and eukaryotic cells. We hope to thereby emphasize the usefulness of in-cell NMR techniques for cellular studies of complex biological processes and for structural analyses in native environments. © 2010 Elsevier Ltd.


Kemmer G.,Leibniz Institute for Molecular Pharmacology | Keller S.,Leibniz Institute for Molecular Pharmacology
Nature Protocols | Year: 2010

We describe an intuitive and rapid procedure for analyzing experimental data by nonlinear least-squares fitting (NLSF) in the most widely used spreadsheet program. Experimental data in x/y form and data calculated from a regression equation are inputted and plotted in a Microsoft Excel worksheet, and the sum of squared residuals is computed and minimized using the Solver add-in to obtain the set of parameter values that best describes the experimental data. The confidence of best-fit values is then visualized and assessed in a generally applicable and easily comprehensible way. Every user familiar with the most basic functions of Excel will be able to implement this protocol, without previous experience in data fitting or programming and without additional costs for specialist software. The application of this tool is exemplified using the well-known Michaelis-Menten equation characterizing simple enzyme kinetics. Only slight modifications are required to adapt the protocol to virtually any other kind of dataset or regression equation. The entire protocol takes 1 h. © 2010 Nature Publishing Group.


Daumke O.,Max Delbruck Centrum fur Molekulare Medizin | Daumke O.,Free University of Berlin | Roux A.,University of Geneva | Haucke V.,Leibniz Institute for Molecular Pharmacology | Haucke V.,Charité - Medical University of Berlin
Cell | Year: 2014

Biological membranes undergo constant remodeling by membrane fission and fusion to change their shape and to exchange material between subcellular compartments. During clathrin-mediated endocytosis, the dynamic assembly and disassembly of protein scaffolds comprising members of the bin-amphiphysin-rvs (BAR) domain protein superfamily constrain the membrane into distinct shapes as the pathway progresses toward fission by the GTPase dynamin. In this Review, we discuss how BAR domain protein assembly and disassembly are controlled in space and time and which structural and biochemical features allow the tight regulation of their shape and function to enable dynamin-mediated membrane fission. © 2014 Elsevier Inc.


Wieffer M.,Leibniz Institute for Molecular Pharmacology
Current biology : CB | Year: 2013

Endosomal membrane traffic serves crucial roles in cell physiology, signaling, and development. Sorting between endosomes and the trans-Golgi network (TGN) is regulated among other factors by the adaptor AP-1, an essential component of multicellular organisms. Membrane recruitment of AP-1 requires phosphatidylinositol 4-phosphate [PI(4)P], though the precise mechanisms and PI4 kinase isozyme (or isozymes) involved in generation of this PI(4)P pool remain unclear. The Wnt pathway is a major developmental signaling cascade and depends on endosomal sorting in Wnt-sending cells. Whether TGN/endosomal sorting modulates signaling downstream of Frizzled (Fz) receptors in Wnt-receiving cells is unknown. Here, we identify PI4-kinase type 2β (PI4K2β) as a regulator of TGN/endosomal sorting and Wnt signaling. PI4K2β and AP-1 interact directly and are required for efficient sorting between endosomes and the TGN. Zebrafish embryos depleted of PI4K2β or AP-1 lack pectoral fins due to defective Wnt signaling. Rescue experiments demonstrate requirements for PI4K2β-AP-1 complex formation and PI4K2β-mediated PI(4)P synthesis. Furthermore, PI4K2β binds to the Fz-associated component Dishevelled (Dvl) and regulates endosomal recycling of Fz receptors and Wnt target gene expression. These data reveal an evolutionarily conserved role for PI4K2β and AP-1 in coupling phosphoinositide metabolism to AP-1-mediated sorting and Wnt signaling. Copyright © 2013 Elsevier Ltd. All rights reserved.


Stauber T.,Leibniz Institute for Molecular Pharmacology | Jentsch T.J.,Leibniz Institute for Molecular Pharmacology
Annual Review of Physiology | Year: 2013

Luminal acidification is of pivotal importance for the physiology of the secretory and endocytic pathways and its diverse trafficking events. Acidification by the proton-pumping V-ATPase requires charge compensation by counterion currents that are commonly attributed to chloride. The molecular identification of intracellular chloride transporters and the improvement of methodologies for measuring intraorganellar pH and chloride have facilitated the investigation of the physiology of vesicular chloride transport. New data question the requirement of chloride for pH regulation of various organelles and furthermore ascribe functions to chloride that are beyond merely electrically shunting the proton pump. This review surveys the currently established and proposed intracellular chloride transporters and gives an overview of membrane-trafficking steps that are affected by the perturbation of chloride transport. Finally, potential mechanisms of membrane-trafficking modulation by chloride are discussed and put into the context of organellar ion homeostasis in general. Copyright © 2013 by Annual Reviews. All rights reserved.


Schroder L.,Leibniz Institute for Molecular Pharmacology
Physica Medica | Year: 2013

NMR studies with hyperpolarized xenon as functionalized sensor or contrast agent recently made notable progress in developing a new approach for detecting molecular markers and parameters of biomedical interest. Combining spin polarization enhancement with novel indirect detection schemes easily enables a 107-fold signal gain, thus having promising potential to solve the NMR sensitivity problem in many applications. Though an inert element, 129Xe has exquisite NMR properties to sense molecular environments. This review summarizes recent developments in the production of hyperpolarized xenon and the design and detection schemes of xenon biosensors. © 2011 Associazione Italiana di Fisica Medica.

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