FMP Publications

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

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Automatic (1)H-NMR Screening of Fatty Acid Composition in Edible Oils
Castejon(*), D., Fricke, P., Cambero(*), M. I.; Herrera(*), A.
Nutrients, 8:93

Tags: Molecular Biophysics (Lange, A.)

Abstract: In this work, we introduce an NMR-based screening method for the fatty acid composition analysis of edible oils. We describe the evaluation and optimization needed for the automated analysis of vegetable oils by low-field NMR to obtain the fatty acid composition (FAC). To achieve this, two scripts, which automatically analyze and interpret the spectral data, were developed. The objective of this work was to drive forward the automated analysis of the FAC by NMR. Due to the fact that this protocol can be carried out at low field and that the complete process from sample preparation to printing the report only takes about 3 min, this approach is promising to become a fundamental technique for high-throughput screening. To demonstrate the applicability of this method, the fatty acid composition of extra virgin olive oils from various Spanish olive varieties (arbequina, cornicabra, hojiblanca, manzanilla, and picual) was determined by (1)H-NMR spectroscopy according to this protocol.

High resolution observed in 800 MHz DNP spectra of extremely rigid type III secretion needles
Fricke, P., Mance(*), D., Chevelkov, V., Giller(*), K., Becker(*), S., Baldus(*), M.; Lange, A.
J Biomol NMR, 65:121-126

Tags: Molecular Biophysics (Lange, A.)

Abstract: The cryogenic temperatures at which dynamic nuclear polarization (DNP) solid-state NMR experiments need to be carried out cause line-broadening, an effect that is especially detrimental for crowded protein spectra. By increasing the magnetic field strength from 600 to 800 MHz, the resolution of DNP spectra of type III secretion needles (T3SS) could be improved by 22 %, indicating that inhomogeneous broadening is not the dominant effect that limits the resolution of T3SS needles under DNP conditions. The outstanding spectral resolution of this system under DNP conditions can be attributed to its low overall flexibility.

Surface Binding of TOTAPOL Assists Structural Investigations of Amyloid Fibrils by Dynamic Nuclear Polarization NMR Spectroscopy
Nagaraj, M., Franks, T. W., Saeidpour(*), S., Schubeis(*), T., Oschkinat, H., Ritter(*), C.; van Rossum, B. J.
Chembiochem, 17:1308-1311

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Dynamic nuclear polarization (DNP) NMR can enhance sensitivity but often comes at the price of a substantial loss of resolution. Two major factors affect spectral quality: low-temperature heterogeneous line broadening and paramagnetic relaxation enhancement (PRE) effects. Investigations by NMR spectroscopy, isothermal titration calorimetry (ITC), and EPR revealed a new substantial affinity of TOTAPOL to amyloid surfaces, very similar to that shown by the fluorescent dye thioflavin-T (ThT). As a consequence, DNP spectra with remarkably good resolution and still reasonable enhancement could be obtained at very low TOTAPOL concentrations, typically 400 times lower than commonly employed. These spectra yielded several long-range constraints that were difficult to obtain without DNP. Our findings open up new strategies for structural studies with DNP NMR spectroscopy on amyloids that can bind the biradical with affinity similar to that shown towards ThT.

Reversible Opening of Intercellular Junctions of Intestinal Epithelial and Brain Endothelial Cells With Tight Junction Modulator Peptides
Bocsik(*), A., Walter(*), F. R., Gyebrovszki(*), A., Fulop(*), L., Blasig, I., Dabrowski, S., Otvos(*), F., Toth(*), A., Rakhely(*), G., Veszelka(*), S., Vastag(*), M., Szabo-Revesz(*), P.; Deli(*), M. A.
Journal of pharmaceutical sciences, 105:754-765

Tags: Molecular Cell Physiology (Blasig, I.E.)

Abstract: The intercellular junctions restrict the free passage of hydrophilic compounds through the paracellular clefts. Reversible opening of the tight junctions of biological barriers is investigated as one of the ways to increase drug delivery to the systemic circulation or the central nervous system. Six peptides, ADT-6, HAV-6, C-CPE, 7-mer (FDFWITP, PN-78), AT-1002, and PN-159, acting on different integral membrane and linker junctional proteins were tested on Caco-2 intestinal epithelial cell line and a coculture model of the blood-brain barrier. All peptides tested in nontoxic concentrations showed a reversible tight junctions modulating effect and were effective to open the paracellular pathway for the marker molecules fluorescein and albumin. The change in the structure of cell-cell junctions was verified by immunostaining for occludin, claudin-4,-5, ZO-1, beta-catenin, and E-cadherin. Expression levels of occludin and claudins were measured in both models. We could demonstrate a selectivity of C-CPE, ADT-6, and HAV-6 peptides for epithelial cells and 7-mer and AT-1002 peptides for brain endothelial cells. PN-159 was the most effective modulator of junctional permeability in both models possibly acting via claudin-1 and -5. Our results indicate that these peptides can be effectively and selectively used as potential pharmaceutical excipients to improve drug delivery across biological barriers.

The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6
Boschert(*), V., Frisch(*), C., Back(*), J. W., van Pee(*), K., Weidauer(*), S. E., Muth(*), E. M., Schmieder, P., Beerbaum, M., Knappik(*), A., Timmerman(*), P.; Mueller(*), T. D.
Open biology, 6

Tags: Solution NMR (Schmieder)

Abstract: The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure-function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis.

Chemical tools for interrogating inositol pyrophosphate structure and function
Brown, N. W., Marmelstein, A. M.; Fiedler, D.
Chem Soc Rev, 45:6311-6326

Tags: Chemical Biology I (Fiedler)

Abstract: The inositol pyrophosphates (PP-InsPs) are a unique group of intracellular messengers that represent some of the most highly phosphorylated molecules in nature. Genetic perturbation of the PP-InsP biosynthetic network indicates a central role for these metabolites in maintaining cellular energy homeostasis and in controlling signal transduction networks. However, despite their discovery over two decades ago, elucidating their physiologically relevant isomers, the biochemical pathways connecting these molecules to their associated phenotypes, and their modes of signal transduction has often been stymied by technical challenges. Many of the advances in understanding these molecules to date have been facilitated by the total synthesis of the various PP-InsP isomers and by the development of new methods that are capable of identifying their downstream signalling partners. Chemical tools have also been developed to distinguish between the proposed PP-InsP signal transduction mechanisms: protein binding, and a covalent modification of proteins termed protein pyrophosphorylation. In this article, we review these recent developments, discuss how they have helped to illuminate PP-InsP structure and function, and highlight opportunities for future discovery.

Cellular Cations Control Conformational Switching of Inositol Pyrophosphate Analogues
Hager(*), A., Wu(*), M., Wang(*), H., Brown, N. W., Jr., Shears(*), S. B., Veiga(*), N.; Fiedler, D.
Chemistry, 22:12406-12414

Tags: Chemical Biology I (Fiedler)

Abstract: The inositol pyrophosphate messengers (PP-InsPs) are emerging as an important class of cellular regulators. These molecules have been linked to numerous biological processes, including insulin secretion and cancer cell migration, but how they trigger such a wide range of cellular responses has remained unanswered in many cases. Here, we show that the PP-InsPs exhibit complex speciation behaviour and propose that a unique conformational switching mechanism could contribute to their multifunctional effects. We synthesised non-hydrolysable bisphosphonate analogues and crystallised the analogues in complex with mammalian PPIP5K2 kinase. Subsequently, the bisphosphonate analogues were used to investigate the protonation sequence, metal-coordination properties, and conformation in solution. Remarkably, the presence of potassium and magnesium ions enabled the analogues to adopt two different conformations near physiological pH. Understanding how the intrinsic chemical properties of the PP-InsPs can contribute to their complex signalling outputs will be essential to elucidate their regulatory functions.

Inositol polyphosphates intersect with signaling and metabolic networks via two distinct mechanisms
Wu(*), M., Chong, L. S., Perlman(*), D. H., Resnick(*), A. C.; Fiedler, D.
Proc Natl Acad Sci U S A, 113:E6757-E6765

Tags: Chemical Biology I (Fiedler)

Abstract: Inositol-based signaling molecules are central eukaryotic messengers and include the highly phosphorylated, diffusible inositol polyphosphates (InsPs) and inositol pyrophosphates (PP-InsPs). Despite the essential cellular regulatory functions of InsPs and PP-InsPs (including telomere maintenance, phosphate sensing, cell migration, and insulin secretion), the majority of their protein targets remain unknown. Here, the development of InsP and PP-InsP affinity reagents is described to comprehensively annotate the interactome of these messenger molecules. By using the reagents as bait, >150 putative protein targets were discovered from a eukaryotic cell lysate (Saccharomyces cerevisiae). Gene Ontology analysis of the binding partners revealed a significant overrepresentation of proteins involved in nucleotide metabolism, glucose metabolism, ribosome biogenesis, and phosphorylation-based signal transduction pathways. Notably, we isolated and characterized additional substrates of protein pyrophosphorylation, a unique posttranslational modification mediated by the PP-InsPs. Our findings not only demonstrate that the PP-InsPs provide a central line of communication between signaling and metabolic networks, but also highlight the unusual ability of these molecules to access two distinct modes of action.

A Stable Pyrophosphoserine Analog for Incorporation into Peptides and Proteins
Yates(*), L. M.; Fiedler, D.
ACS Chem Biol, 11:1066-1073

Tags: Chemical Biology I (Fiedler)

Abstract: Protein pyrophosphorylation is a covalent modification of proteins, mediated by the inositol pyrophosphate messengers. Although the inositol pyrophosphates have been linked to a range of cellular processes, the role of protein pyrophosphorylation remains minimally characterized in vivo. The inherent instability of the phosphoanhydride bond has hampered the development of useful bioanalytical techniques to interrogate this novel signaling mechanism. Here, we describe the preparation of a pyrophosphoserine analog containing a stable methylene-bisphosphonate group that is compatible with solid-phase peptide synthesis. The resulting peptides demonstrate enhanced stability in Eukaryotic cell lysates and mammalian plasma and display resistance toward chemical degradation, when compared to the corresponding pyrophosphopeptides. In addition, the peptides containing the stable pyrophosphoserine analog are highly compatible with common ligation methods, such as native chemical ligation, maleimide conjugation, and glutaraldehyde ligation. The bisphosphonate-containing peptides will, therefore, be well-suited for future pyrophosphoserine antibody generation and affinity capture of pyrophosphoprotein binding partners and provide a key entry point to study the regulatory role of protein pyrophosphorylation.

Elm defence against herbivores and pathogens: morphological, chemical and molecular regulation aspects
Buchel(*), K., Fenning(*), T., Gershenzon(*), J., Hilker(*), M.; Meiners, T.
Phytochem Rev, 15:961-983

Tags: Department Chemical Biology/ EU-OPENSCREEN

Abstract: Elms (Ulmus spp.) have long been appreciated for their environmental tolerance, landscape and ornamental value, and the quality of their wood. Although elm trees are extremely hardy against abiotic stresses such as wind and pollution, they are susceptible to attacks of biotic stressors. Over 100 phytopathogens and invertebrate pests are associated with elms: fungi, bacteria and insects like beetles and moths, and to a lesser extent aphids, mites, viruses and nematodes. While the biology of the pathogen and insect vector of the Dutch elm disease has been intensively studied, less attention has been paid so far to the defence mechanisms of elms to other biotic stressors. This review highlights knowledge of direct and indirect elm defences against biotic stressors focusing on morphological, chemical and gene regulation aspects. First, we report how morphological defence mechanisms via barrier formation and vessel occlusion prevent colonisation and spread of wood- and bark-inhabiting fungi and bacteria. Second, we outline how secondary metabolites such as terpenoids (volatile terpenoids, mansonones and triterpenoids) and phenolics (lignans, coumarins, flavonoids) in leaves and bark are involved in constitutive and induced chemical defence mechanisms of elms. Third, we address knowledge on how the molecular regulation of elm defence is orchestrated through the interaction of a huge variety of stress- and defence-related genes. We conclude by pointing to the gaps of knowledge on the chemical and molecular mechanisms of elm defence against pest insects and diseases. An in-depth understanding of defence mechanisms of elms will support the development of sustainable integrated management of pests and diseases attacking elms.

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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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