FMP Publications

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

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References
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
(2017)

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.

Chemical Approaches to Studying Labile Amino Acid Phosphorylation
Marmelstein, A. M., Moreno, J.; Fiedler, D.
Topics in current chemistry (Journal), 375:22
(2017)

Tags: Chemical Biology I (Fiedler)

Abstract: Phosphorylation of serine, threonine, and tyrosine residues is the archetypal posttranslational modification of proteins. While phosphorylation of these residues has become standard textbook knowledge, phosphorylation of other amino acid side chains is underappreciated and minimally characterized by comparison. This disparity is rooted in the relative instability of these chemically distinct amino acid side chain moieties, namely phosphoramidates, acyl phosphates, thiophosphates, and phosphoanhydrides. In the case of the O-phosphorylated amino acids, synthetic constructs were critical to assessing their stability and developing tools for their study. As the chemical biology community has become more aware of these alternative phosphorylation sites, methodology has been developed for the synthesis of well-characterized standards and close mimics of these phosphorylated amino acids as well. In this article, we review the synthetic chemistry that is a prerequisite to progress in this field.

An Integrative Framework Reveals Signaling-to-Transcription Events in Toll-like Receptor Signaling
Mertins(*), P., Przybylski(*), D., Yosef(*), N., Qiao(*), J., Clauser(*), K., Raychowdhury(*), R., Eisenhaure(*), T. M., Maritzen, T., Haucke, V., Satoh(*), T., Akira(*), S., Carr(*), S. A., Regev(*), A., Hacohen(*), N.; Chevrier(*), N.
Cell Rep, 19:2853-2866
(2017)

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

Abstract: Building an integrated view of cellular responses to environmental cues remains a fundamental challenge due to the complexity of intracellular networks in mammalian cells. Here, we introduce an integrative biochemical and genetic framework to dissect signal transduction events using multiple data types and, in particular, to unify signaling and transcriptional networks. Using the Toll-like receptor (TLR) system as a model cellular response, we generate multifaceted datasets on physical, enzymatic, and functional interactions and integrate these data to reveal biochemical paths that connect TLR4 signaling to transcription. We define the roles of proximal TLR4 kinases, identify and functionally test two dozen candidate regulators, and demonstrate a role for Ap1ar (encoding the Gadkin protein) and its binding partner, Picalm, potentially linking vesicle transport with pro-inflammatory responses. Our study thus demonstrates how deciphering dynamic cellular responses by integrating datasets on various regulatory layers defines key components and higher-order logic underlying signaling-to-transcription pathways.

Heparan Sulfates Support Pyramidal Cell Excitability, Synaptic Plasticity, and Context Discrimination
Minge(*), D., Senkov(*), O., Kaushik(*), R., Herde(*), M. K., Tikhobrazova(*), O., Wulff(*), A. B., Mironov(*), A., van Kuppevelt(*), T. H., Oosterhof(*), A., Kochlamazashvili, G., Dityatev(*), A.; Henneberger(*), C.
Cerebral cortex (New York, N.Y. : 1991), 27:903-918
(2017)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Heparan sulfate (HS) proteoglycans represent a major component of the extracellular matrix and are critical for brain development. However, their function in the mature brain remains to be characterized. Here, acute enzymatic digestion of HS side chains was used to uncover how HSs support hippocampal function in vitro and in vivo. We found that long-term potentiation (LTP) of synaptic transmission at CA3-CA1 Schaffer collateral synapses was impaired after removal of highly sulfated HSs with heparinase 1. This reduction was associated with decreased Ca2+ influx during LTP induction, which was the consequence of a reduced excitability of CA1 pyramidal neurons. At the subcellular level, heparinase treatment resulted in reorganization of the distal axon initial segment, as detected by a reduction in ankyrin G expression. In vivo, digestion of HSs impaired context discrimination in a fear conditioning paradigm and oscillatory network activity in the low theta band after fear conditioning. Thus, HSs maintain neuronal excitability and, as a consequence, support synaptic plasticity and learning.

Structure of the competence pilus major pilin ComGC in Streptococcus pneumoniae
Muschiol(*), S., Erlendsson(*), S., Aschtgen(*), M. S., Oliveira(*), V., Schmieder, P., de Lichtenberg(*), C., Teilum(*), K., Boesen(*), T., Akbey(*), Ü.; Henriques-Normark(*), B.
J Biol Chem, 292:14134-14146
(2017)

Tags: Solution NMR (Schmieder)

Abstract: Type IV pili are important virulence factors on the surface of many pathogenic bacteria and have been implicated in a wide range of diverse functions, including attachment, twitching motility, biofilm formation, and horizontal gene transfer. The respiratory pathogen Streptococcus pneumoniae deploys type IV pili to take up DNA during transformation. These "competence pili" are composed of the major pilin protein ComGC and exclusively assembled during bacterial competence, but their biogenesis remains unclear. Here, we report the high resolution NMR structure of N-terminal truncated ComGC revealing a highly flexible and structurally divergent type IV pilin. It consists of only three alpha-helical segments forming a well-defined electronegative cavity and confined electronegative and hydrophobic patches. The structure is particularly flexible between the first and second alpha-helix with the first helical part exhibiting slightly slower dynamics than the rest of the pilin, suggesting that the first helix is involved in forming the pilus structure core and that parts of helices two and three are primarily surface-exposed. Taken together, our results provide the first structure of a type IV pilin protein involved in the formation of competence-induced pili in Gram-positive bacteria and corroborate the remarkable structural diversity among type IV pilin proteins.

Loss of the Na+/H+ exchanger NHE8 causes male infertility in mice by disrupting acrosome formation
Oberheide, K., Puchkov, D.; Jentsch, T. J.
J Biol Chem,
(2017)

Tags: Physiology and Pathology of Ion Transport (Jentsch), Cellular Imaging (Wiesner/Puchkov)

Abstract: Mammalian sperm feature a specialized secretory organelle on the anterior part of the sperm nucleus, the acrosome, which is essential for male fertility. It is formed by a fusion of Golgi-derived vesicles. We show here that the predominantly Golgi-resident Na+/H+ exchanger NHE8 localizes to the developing acrosome of spermatids. Similar to wild-type mice, Nhe8-/- mice generated Golgi-derived vesicles positive for acrosomal markers and attached to nuclei, but these vesicles failed to form large acrosomal granules and the acrosomal cap. Spermatozoa from Nhe8-/- mice completely lacked acrosomes, were round-headed, exhibited abnormal mitochondrial distribution and displayed decreased motility, resulting in selective male infertility. Of note, similar features are also found in globozoospermia, one of the causes of male infertility in humans. Germ cell-specific, but not Sertoli cell-specific Nhe8 disruption recapitulated the globozoospermia phenotype, demonstrating that NHE8's role in spermiogenesis is germ cell-intrinsic. Our work has uncovered a crucial role of NHE8 in acrosome biogenesis and suggests that some forms of human globozoospermia might be caused by a loss of function of this Na+/H+ exchanger. It points to NHE8 as a candidate gene for human globozoospermia and a possible drug target for male contraception.

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.

Unambiguous Identification of Serine and Threonine Pyrophosphorylation Using Neutral-Loss-Triggered Electron-Transfer/Higher-Energy Collision Dissociation
Penkert, M., Yates(*), L. M., Schümann, M., Perlman(*), D., Fiedler, D.; Krause, E.
Anal Chem, 89:3672-3680
(2017)

Tags: Mass Spectrometry (Krause, E.), Chemical Biology I (Fiedler)

Abstract: Tandem mass spectrometry (MS/MS) has emerged as the core technology for identification of post-translational modifications (PTMs). Here, we report the mass spectrometry analysis of serine and threonine pyrophosphorylation, a protein modification that has eluded detection by conventional MS/MS methods. Analysis of a set of synthesized, site-specifically modified peptides by different fragmentation techniques shows that pyrophosphorylated peptides exhibit a characteristic neutral loss pattern of 98, 178, and 196 Da, which enables the distinction between isobaric pyro- and diphosphorylated peptides. In addition, electron-transfer dissociation combined with higher energy collision dissociation (EThcD) provides exceptional data-rich MS/MS spectra for direct and unambiguous pyrophosphosite assignment. Remarkably, sufficient fragmentation of doubly charged precursors could be achieved by electron-transfer dissociation (ETD) with increased supplemental activation, without losing the labile modification. By exploiting the specific fragmentation behavior of pyrophosphorylated peptides during collision-induced dissociation (CID), a data dependent neutral-loss-triggered EThcD acquisition method was developed. This strategy enables reliable pyrophosphopeptide identification in complex samples, without compromising speed and sensitivity.

Polar and charged extracellular residues conserved among barrier-forming claudins contribute to tight junction strand formation
Piontek, A., Rossa, J., Protze, J., Wolburg(*), H., Hempel(*), C., Günzel(*), D., Krause, G.; Piontek(*), J.
Annals of the New York Academy of Sciences,
(2017)

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

Abstract: Claudins (Cldn) form the backbone of tight junction (TJ) strands and thereby regulate paracellular permeability for solutes and water. Polymeric strands are formed by homo- and heterophilic cis- and trans-interactions between claudin protomers. Crystal structures of some claudins have been resolved; however, the mechanism by which claudins assemble into TJ strands remains unclear. To elucidate strand architecture, TJ-like strands were reconstituted in HEK293 cells by claudin transfection. Determinants of prototypic, classic barrier-forming claudins (Cldn1, -3, and -5) involved in strand formation were analyzed by mutagenesis. The capability of claudin constructs to interact in trans and to form strands was investigated by cell contact-enrichment assays and freeze-fracture electron microscopy. Residues in extracellular loops 1 and 2 of the claudins affecting strand formation were identified. Using homology modeling and molecular docking, we tested working concepts for the arrangement of claudin protomers within TJ strands. We show that the charge of Lys65 in Cldn1 and Glu158 in Cldn3, but not of Arg30 or Asp145 in Cldn3, and the polarity of Gln56 and Gln62 in Cldn3 and of Gln57 in Cldn5 are necessary for TJ strand formation. These residues are all conserved among barrier-forming classic claudins. The results contribute to mechanistic understanding of claudin-based regulation of paracellular permeability.

A cCPE-based xenon biosensor for magnetic resonance imaging of claudin-expressing cells
Piontek, A., Witte, C., May Rose, H., Eichner(*), M., Protze, J., Krause, G., Piontek(*), J.; Schröder, L.
Annals of the New York Academy of Sciences, 1397:195-208
(2017)

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

Abstract: The majority of malignant tumors originate from epithelial cells, and many of them are characterized by an overexpression of claudins (Cldns) and their mislocalization out of tight junctions. We utilized the C-terminal claudin-binding domain of Clostridium perfringens enterotoxin (cCPE), with its high affinity to specific members of the claudin family, as the targeting unit for a claudin-sensitive cancer biosensor. To overcome the poor sensitivity of conventional relaxivity-based magnetic resonance imaging (MRI) contrast agents, we utilized the superior sensitivity of xenon Hyper-CEST biosensors. We labeled cCPE for both xenon MRI and fluorescence detection. As one readout module, we employed a cryptophane (CrA) monoacid and, as the second, a fluorescein molecule. Both were conjugated separately to a biotin molecule via a polyethyleneglycol chemical spacer and later via avidin linked to GST-cCPE. Nontransfected HEK293 cells and HEK293 cells stably expressing Cldn4-FLAG were incubated with the cCPE-based biosensor. Fluorescence-based flow cytometry and xenon MRI demonstrated binding of the biosensor specifically to Cldn4-expressing cells. This study provides proof of concept for the use of cCPE as a carrier for diagnostic contrast agents, a novel approach for potential detection of Cldn3/-4-overexpressing tumors for noninvasive early cancer detection.

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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 
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info(at)fmp-berlin.de

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