FMP Publications

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

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References
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.

Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS
Leonhardt(*), J., Villela(*), D. C., Teichmann, A., Munter(*), L. M., Mayer(*), M. C., Mardahl(*), M., Kirsch(*), S., Namsolleck(*), P., Lucht(*), K., Benz(*), V., Alenina(*), N., Daniell(*), N., Horiuchi(*), M., Iwai(*), M., Multhaup(*), G., Schülein, R., Bader(*), M., Santos(*), R. A., Unger(*), T.; Steckelings(*), U. M.
Hypertension,
(2017)

Tags: Protein Trafficking (Schülein), Cellular Imaging (Wiesner)

Abstract: The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin-angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa. Such cross-inhibition may indicate heterodimerization of these receptors. Therefore, this study investigated the molecular and functional interplay between MAS and the AT2R. Molecular interactions were assessed by fluorescence resonance energy transfer and by cross correlation spectroscopy in human embryonic kidney-293 cells transfected with vectors encoding fluorophore-tagged MAS or AT2R. Functional interaction of AT2R and MAS was studied in astrocytes with CX3C chemokine receptor-1 messenger RNA expression as readout. Coexpression of fluorophore-tagged AT2R and MAS resulted in a fluorescence resonance energy transfer efficiency of 10.8 +/- 0.8%, indicating that AT2R and MAS are capable to form heterodimers. Heterodimerization was verified by competition experiments using untagged AT2R and MAS. Specificity of dimerization of AT2R and MAS was supported by lack of dimerization with the transient receptor potential cation channel, subfamily C-member 6. Dimerization of the AT2R was abolished when it was mutated at cysteine residue 35. AT2R and MAS stimulation with the respective agonists, Compound 21 or angiotensin-(1-7), significantly induced CX3C chemokine receptor-1 messenger RNA expression. Effects of each agonist were blocked by an AT2R antagonist (PD123319) and also by a MAS antagonist (A-779). Knockout of a single of these receptors made astrocytes unresponsive for both agonists. Our results suggest that MAS and the AT2R form heterodimers and that-at least in astrocytes-both receptors functionally depend on each other.

The complex co-translational processing of glycoprotein GP5 of type 1 porcine reproductive and respiratory syndrome virus
Thaa(*), B., Kaufer(*), S., Neumann(*), S. A., Peibst(*), B., Nauwynck(*), H., Krause, E.; Veit(*), M.
Virus Res, 240:112-120
(2017)

Tags: Mass Spectrometry (Krause, E.)

Abstract: GP5 and M, the major membrane proteins of porcine reproductive and respiratory syndrome virus (PRRSV), are the driving force for virus budding and a target for antibodies. We studied co-translational processing of GP5 from an European PRRSV-1 strain. Using mass spectrometry, we show that in virus particles of a Lelystad variant, the signal peptide of GP5 was absent due to cleavage between glycine-34 and asparagine-35. This cleavage site removes an epitope for a neutralizing monoclonal antibody, but leaves intact another epitope recognized by neutralizing pig sera. Upon ectopic expression of this GP5 in cells, signal peptide cleavage was however inefficient. Complete cleavage occurred when cysteine-24 was changed to proline or an unused glycosylation site involving asparagine-35 was mutated. Insertion of proline at position 24 also caused carbohydrate attachment to asparagine-35. Glycosylation sites introduced downstream of residue 35 were used, but did not inhibit signal peptide processing. Co-expression of the M protein rescued this processing defect in GP5, suggesting a novel function of M towards GP5. We speculate that a complex interplay of the co-translational modifications of GP5 affect the N-terminal structure of the mature proteins and hence its antigenicity.

Helical Polyisocyanopeptides as Lyotropic Liquid Crystals for Measuring Residual Dipolar Couplings
Li(*), G. W., Cao(*), J. M., Zong(*), W., Hu(*), L., Hu(*), M. L., Lei(*), X., Sun, H.; Tan(*), R. X.
Chemistry, 23:7653-7656
(2017)

Tags: Computational Chemistry and Protein Design (Kühne)

Abstract: Residual dipolar couplings (RDC) emerged to be an important structural parameter for organic and biomolecules. Herein, a new helical polyisocyanopeptide (l,l-PIAF-OBn) that forms lyotropic liquid crystals (LLC) in CDCl3 is proposed as a novel weakly orienting medium for acquiring residual dipolar couplings (RDCs) of organic molecules. We demonstrate its application for the structural elucidation of strychnine and triptolide.

NMR Hyperpolarization Techniques of Gases
Barskiy(*), D. A., Coffey(*), A. M., Nikolaou(*), P., Mikhaylov(*), D. M., Goodson(*), B. M., Branca(*), R. T., Lu(*), G. J., Shapiro(*), M. G., Telkki(*), V. V., Zhivonitko(*), V. V., Koptyug(*), I. V., Salnikov(*), O. G., Kovtunov(*), K. V., Bukhtiyarov(*), V. I., Rosen(*), M. S., Barlow(*), M. J., Safavi(*), S., Hall(*), I. P., Schroeder, L.; Chekmenev(*), E. Y.
Chemistry, 23:725-751
(2017)

Tags: Molecular Imaging (Schröder)

Abstract: Nuclear spin polarization can be significantly increased through the process of hyperpolarization, leading to an increase in the sensitivity of nuclear magnetic resonance (NMR) experiments by 4-8 orders of magnitude. Hyperpolarized gases, unlike liquids and solids, can often be readily separated and purified from the compounds used to mediate the hyperpolarization processes. These pure hyperpolarized gases enabled many novel MRI applications including the visualization of void spaces, imaging of lung function, and remote detection. Additionally, hyperpolarized gases can be dissolved in liquids and can be used as sensitive molecular probes and reporters. This Minireview covers the fundamentals of the preparation of hyperpolarized gases and focuses on selected applications of interest to biomedicine and materials science.

Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR
Erlendsson(*), S., Gotfryd(*), K., Larsen, F. H., Mortensen(*), J. S., Geiger, M. A., van Rossum, B. J., Oschkinat, H., Gether(*), U., Teilum(*), K.; Loland(*), C. J.
Elife, 6
(2017)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT. We were able to produce high-quality NMR spectra of substrate bound to microcrystalline LeuT samples and identify one set of sodium-dependent substrate-specific chemical shifts. Furthermore, our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein. We conclude that under our experimental conditions there is only one detectable leucine molecule bound to LeuT.

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.

Design of S-Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl-tRNA Synthetase Variant
Exner(*), M. P., Kuenzl(*), T., To(*), T. M., Ouyang(*), Z., Schwagerus, S., Hoesl(*), M. G., Hackenberger, C. P., Lensen(*), M. C., Panke(*), S.; Budisa(*), N.
Chembiochem, 18:85-90
(2017)

Tags: Chemical Biology II (Hackenberger)

Abstract: The noncanonical amino acid S-allyl cysteine (Sac) is one of the major compounds of garlic extract and exhibits a range of biological activities. It is also a small bioorthogonal alkene tag capable of undergoing controlled chemical modifications, such as photoinduced thiol-ene coupling or Pd-mediated deprotection. Its small size guarantees minimal interference with protein structure and function. Here, we report a simple protocol efficiently to couple in-situ semisynthetic biosynthesis of Sac and its incorporation into proteins in response to amber (UAG) stop codons. We exploited the exceptional malleability of pyrrolysyl-tRNA synthetase (PylRS) and evolved an S-allylcysteinyl-tRNA synthetase (SacRS) capable of specifically accepting the small, polar amino acid instead of its long and bulky aliphatic natural substrate. We succeeded in generating a novel and inexpensive strategy for the incorporation of a functionally versatile amino acid. This will help in the conversion of orthogonal translation from a standard technique in academic research to industrial biotechnology.

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.

Structural Basis of the Oncogenic Interaction of Phosphatase PRL-1 with the Magnesium Transporter CNNM2
Gimenez-Mascarell(*), P., Oyenarte(*), I., Hardy(*), S., Breiderhoff(*), T., Stuiver, M., Kostantin(*), E., Diercks(*), T., Pey(*), A. L., Ereno-Orbea(*), J., Martinez-Chantar(*), M. L., Khalaf-Nazzal(*), R., Claverie-Martin(*), F., Müller(*), D., Tremblay(*), M. L.; Martinez-Cruz(*), L. A.
J Biol Chem, 292:786-801
(2017)

Tags: In-Cell NMR (Selenko)

Abstract: Phosphatases of regenerating liver (PRLs), the most oncogenic of all protein-tyrosine phosphatases (PTPs), play a critical role in metastatic progression of cancers. Recent findings established a new paradigm by uncovering that their association with magnesium transporters of the cyclin M (CNNM) family causes a rise in intracellular magnesium levels that promote oncogenic transformation. Recently, however, essential roles for regulation of the circadian rhythm and reproduction of the CNNM family have been highlighted. Here, we describe the crystal structure of PRL-1 in complex with the Bateman module of CNNM2 (CNNM2BAT), which consists of two cystathionine beta-synthase (CBS) domains (IPR000664) and represents an intracellular regulatory module of the transporter. The structure reveals a heterotetrameric association, consisting of a disc-like homodimer of CNNM2BAT bound to two independent PRL-1 molecules, each one located at opposite tips of the disc. The structure highlights the key role played by Asp-558 at the extended loop of the CBS2 motif of CNNM2 in maintaining the association between the two proteins and proves that the interaction between CNNM2 and PRL-1 occurs via the catalytic domain of the phosphatase. Our data shed new light on the structural basis underlying the interaction between PRL phosphatases and CNNM transporters and provides a hypothesis about the molecular mechanism by which PRL-1, upon binding to CNNM2, might increase the intracellular concentration of Mg2+ thereby contributing to tumor progression and metastasis. The availability of this structure sets the basis for the rational design of compounds modulating PRL-1 and CNNM2 activities.

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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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