FMP Publications

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

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References
Chemical Approaches to Investigate Labile Peptide and Protein Phosphorylation
Hauser, A., Penkert, M.; Hackenberger, C. P. R.
Accounts of chemical research, 50:1883-1893
(2017)

Tags: Chemical Biology II (Hackenberger)

Abstract: Protein phosphorylation is by far the most abundant and most studied post-translational modification (PTM). For a long time, phosphate monoesters of serine (pSer), threonine (pThr), and tyrosine (pTyr) have been considered as the only relevant forms of phosphorylation in organisms. Recently, several research groups have dedicated their efforts to the investigation of other, less characterized phosphoamino acids as naturally occurring PTMs. Such apparent peculiar phosphorylations include the phosphoramidates of histidine (pHis), arginine (pArg), and lysine (pLys), the phosphorothioate of cysteine (pCys), and the anhydrides of pyrophosphorylated serine (ppSer) and threonine (ppThr). Almost all of these phosphorylated amino acids show higher lability under physiological conditions than those of phosphate monoesters. Furthermore, they are prone to hydrolysis under acidic and sometimes basic conditions as well as at elevated temperatures, which renders their synthetic accessibility and proteomic analysis particularly challenging. In this Account, we illustrate recent chemical approaches to probe the occurrence and function of these labile phosphorylation events. Within these endeavors, the synthesis of site-selectively phosphorylated peptides, in particular in combination with chemoselective phosphorylation strategies, was crucial. With these well-defined standards in hand, the appropriate proteomic mass spectrometry-based analysis protocols for the characterization of labile phosphosites in biological samples could be developed. Another successful approach in this research field includes the design and synthesis of stable analogues of these labile PTMs, which were used for the generation of pHis- and pArg-specific antibodies for the detection and enrichment of endogenous phosphorylated samples. Finally, other selective enrichment techniques are described, which rely for instance on the unique chemical environment of a pyrophosphate or the selective interaction between a phosphoamino acid and its phosphatase. It is worth noting that many of those studies are still in their early stages, which is also reflected in the small number of identified phosphosites compared to that of phosphate monoesters. Thus, many challenges need to be mastered to fully understand the biological role of these poorly characterized and rather uncommon phosphorylations. Taken together, this overview exemplifies recent efforts in a flourishing field of functional proteomic analysis and furthermore manifests the power of modern peptide synthesis to address unmet questions in the life sciences.

The ClC-K2 Chloride Channel Is Critical for Salt Handling in the Distal Nephron
Hennings(*), J. C., Andrini(*), O., Picard(*), N., Paulais(*), M., Huebner(*), A. K., Cayuqueo(*), I. K., Bignon(*), Y., Keck(*), M., Corniere(*), N., Böhm(*), D., Jentsch, T. J., Chambrey(*), R., Teulon(*), J., Hübner(*), C. A.; Eladari(*), D.
Journal of the American Society of Nephrology : JASN, 28:209-217
(2017)

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Chloride transport by the renal tubule is critical for blood pressure (BP), acid-base, and potassium homeostasis. Chloride uptake from the urinary fluid is mediated by various apical transporters, whereas basolateral chloride exit is thought to be mediated by ClC-Ka/K1 and ClC-Kb/K2, two chloride channels from the ClC family, or by KCl cotransporters from the SLC12 gene family. Nevertheless, the localization and role of ClC-K channels is not fully resolved. Because inactivating mutations in ClC-Kb/K2 cause Bartter syndrome, a disease that mimics the effects of the loop diuretic furosemide, ClC-Kb/K2 is assumed to have a critical role in salt handling by the thick ascending limb. To dissect the role of this channel in detail, we generated a mouse model with a targeted disruption of the murine ortholog ClC-K2. Mutant mice developed a Bartter syndrome phenotype, characterized by renal salt loss, marked hypokalemia, and metabolic alkalosis. Patch-clamp analysis of tubules isolated from knockout (KO) mice suggested that ClC-K2 is the main basolateral chloride channel in the thick ascending limb and in the aldosterone-sensitive distal nephron. Accordingly, ClC-K2 KO mice did not exhibit the natriuretic response to furosemide and exhibited a severely blunted response to thiazide. We conclude that ClC-Kb/K2 is critical for salt absorption not only by the thick ascending limb, but also by the distal convoluted tubule.

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.

Sleep & metabolism: The multitasking ability of lateral hypothalamic inhibitory circuitries
Herrera(*), C. G., Ponomarenko, A., Korotkova, T., Burdakov(*), D.; Adamantidis(*), A.
Front Neuroendocrinol, 44:27-34
(2017)

Tags: Behavioral Neurodynamics (Korotkova/Ponomarenko)

Abstract: The anatomical and functional mapping of lateral hypothalamic circuits has been limited by the numerous cell types and complex, yet unclear, connectivity. Recent advances in functional dissection of input-output neurons in the lateral hypothalamus have identified subset of inhibitory cells as crucial modulators of both sleep-wake states and metabolism. Here, we summarize these recent studies and discuss the multi-tasking functions of hypothalamic circuitries in integrating sleep and metabolism in the mammalian brain.

Small Molecules Targeting Human N-Acetylmannosamine Kinase
Hinderlich(*), S., Neuenschwander, M., Wratil(*), P. R., Oder, A., Lisurek, M., Nguyen(*), L. D., von Kries, J. P.; Hackenberger, C. P. R.
Chembiochem,
(2017)

Tags: Chemical Biology II (Hackenberger), Screening Unit ( von Kries)

Abstract: N-Acetylmannosamine kinase (MNK) plays a key role in the biosynthesis of sialic acids and glycosylation of proteins. Sialylated glycoconjugates affect a large number of biological processes, including immune modulation and cancer transformation. In search of effective inhibitors of MNK we applied high-throughput screening of drug-like small molecules. By applying different orthogonal assays for their validation we identified four potential MNK-specific inhibitors with IC50 values in the low-micromolar range. Molecular modelling of the inhibitors into the active site of MNK supports their binding to the sugar or the ATP-binding pocket of the enzyme or both. These compounds are promising for downregulation of the sialic acid content of glycoconjugates and for studying the functional contribution of sialic acids to disease development.

Werner Reutter: A Visionary Pioneer in Molecular Glycobiology
Hinderlich(*), S., Tauber(*), R., Bertozzi(*), C. R.; Hackenberger, C. P. R.
Chembiochem, 18:1141-1145
(2017)

Tags: Chemical Biology II (Hackenberger)

Abstract: A creative pioneer: Werner Reutter (1937-2016) was a scientist who both made fundamental discoveries in glycobiology and reached out to disciplines beyond his core field. Many of his former colleagues and students will remember his desire to exchange research ideas, which ultimately contributed to the birth of new research fields.

Molecular features of the L-type amino acid transporter 2 determine different import and export profiles for thyroid hormones and amino acids
Hinz, K. M., Neef, D., Rutz, C., Furkert, J., Köhrle(*), J., Schülein, R.; Krause, G.
Mol Cell Endocrinol, 443:163-174
(2017)

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

Abstract: The L-type amino acid transporter 2 (LAT2) imports amino acids (AA) and also certain thyroid hormones (TH), e.g. 3,3'-T2 and T3, but not rT3 and T4. We utilized LAT2 mutations (Y130A, N133S, F242W) that increase 3,3'-T2 import and focus here on import and export capacity for AA, T4, T3, BCH and derivatives thereof to delineate molecular features. Transport studies and analysis of competitive inhibition of import by radiolabelled TH and AA were performed in Xenopus laevis oocytes. Only Y130A, a pocket widening mutation, enabled import for T4 and increased it for T3. Mutant F242W showed increased 3,3'-T2 import but no import rates for other TH derivatives. No export was detected for any TH by LAT2-wild type (WT). Mutations Y130A and N133S enabled only the export of 3,3'-T2, while N133S also increased AA export. Thus, distinct molecular LAT2-features determine bidirectional AA transport but only an unidirectional 3,3'-T2 and T3 import.

Neuronal Chemosensation and Osmotic Stress Response Converge in the Regulation of aqp-8 in C. elegans
Igual Gil(*), C., Jarius(*), M., von Kries, J. P.; Rohlfing(*), A. K.
Frontiers in physiology, 8:380
(2017)

Tags: Screening Unit (von Kries)

Abstract: Aquaporins occupy an essential role in sustaining the salt/water balance in various cells types and tissues. Here, we present new insights into aqp-8 expression and regulation in Caenorhabditis elegans. We show, that upon exposure to osmotic stress, aqp-8 exhibits a distinct expression pattern within the excretory cell compared to other C. elegans aquaporins expressed. This expression is correlated to the osmolarity of the surrounding medium and can be activated physiologically by osmotic stress or genetically in mutants with constitutively active osmotic stress response. In addition, we found aqp-8 expression to be constitutively active in the TRPV channel mutant osm-9(ok1677). In a genome-wide RNAi screen we identified additional regulators of aqp-8. Many of these regulators are connected to chemosensation by the amphid neurons, e.g., odr-10 and gpa-6, and act as suppressors of aqp-8 expression. We postulate from our results, that aqp-8 plays an important role in sustaining the salt/water balance during a secondary response to hyper-osmotic stress. Upon its activation aqp-8 promotes vesicle docking to the lumen of the excretory cell and thereby enhances the ability to secrete water and transport osmotic active substances or waste products caused by protein damage. In summary, aqp-8 expression and function is tightly regulated by a network consisting of the osmotic stress response, neuronal chemosensation as well as the response to protein damage. These new insights in maintaining the salt/water balance in C. elegans will help to reveal the complex homeostasis network preserved throughout species.

Intersectin 1 is a component of the Reelin pathway to regulate neuronal migration and synaptic plasticity in the hippocampus
Jakob, B., Kochlamazashvili, G., Jaepel, M., Gauhar(*), A., Bock(*), H. H., Maritzen, T.; Haucke, V.
Proc Natl Acad Sci U S A, 114:5533-5538
(2017)

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

Abstract: Brain development and function depend on the directed and coordinated migration of neurons from proliferative zones to their final position. The secreted glycoprotein Reelin is an important factor directing neuronal migration. Loss of Reelin function results in the severe developmental disorder lissencephaly and is associated with neurological diseases in humans. Reelin signals via the lipoprotein receptors very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), but the exact mechanism by which these receptors control cellular function is poorly understood. We report that loss of the signaling scaffold intersectin 1 (ITSN1) in mice leads to defective neuronal migration and ablates Reelin stimulation of hippocampal long-term potentiation (LTP). Knockout (KO) mice lacking ITSN1 suffer from dispersion of pyramidal neurons and malformation of the radial glial scaffold, akin to the hippocampal lamination defects observed in VLDLR or ApoER2 mutants. ITSN1 genetically interacts with Reelin receptors, as evidenced by the prominent neuronal migration and radial glial defects in hippocampus and cortex seen in double-KO mice lacking ITSN1 and ApoER2. These defects were similar to, albeit less severe than, those observed in Reelin-deficient or VLDLR/ ApoER2 double-KO mice. Molecularly, ITSN1 associates with the VLDLR and its downstream signaling adaptor Dab1 to facilitate Reelin signaling. Collectively, these data identify ITSN1 as a component of Reelin signaling that acts predominantly by facilitating the VLDLR-Dab1 axis to direct neuronal migration in the cortex and hippocampus and to augment synaptic plasticity.

Statin and rottlerin small-molecule inhibitors restrict colon cancer progression and metastasis via MACC1
Juneja(*), M., Kobelt(*), D., Walther(*), W., Voss(*), C., Smith(*), J., Specker, E., Neuenschwander, M., Gohlke(*), B. O., Dahlmann(*), M., Radetzki, S., Preissner(*), R., von Kries, J. P., Schlag(*), P. M.; Stein(*), U.
PLoS biology, 15:e2000784
(2017)

Tags: Screening Unit (von Kries)

Abstract: MACC1 (Metastasis Associated in Colon Cancer 1) is a key driver and prognostic biomarker for cancer progression and metastasis in a large variety of solid tumor types, particularly colorectal cancer (CRC). However, no MACC1 inhibitors have been identified yet. Therefore, we aimed to target MACC1 expression using a luciferase reporter-based high-throughput screening with the ChemBioNet library of more than 30,000 compounds. The small molecules lovastatin and rottlerin emerged as the most potent MACC1 transcriptional inhibitors. They remarkably inhibited MACC1 promoter activity and expression, resulting in reduced cell motility. Lovastatin impaired the binding of the transcription factors c-Jun and Sp1 to the MACC1 promoter, thereby inhibiting MACC1 transcription. Most importantly, in CRC-xenografted mice, lovastatin and rottlerin restricted MACC1 expression and liver metastasis. This is-to the best of our knowledge-the first identification of inhibitors restricting cancer progression and metastasis via the novel target MACC1. This drug repositioning might be of therapeutic value for CRC patients.

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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
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info(at)fmp-berlin.de

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