FMP Publications

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

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References
Modes and mechanisms of synaptic vesicle recycling
Soykan, T., Maritzen, T.; Haucke, V.
Curr Opin Neurobiol, 39:17-23
(2016)

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

Abstract: Neurotransmission requires the recycling of synaptic vesicles (SVs) to replenish the SV pool, clear release sites, and maintain presynaptic integrity. In spite of decades of research the modes and mechanisms of SV recycling remain controversial. The identification of clathrin-independent modes of SV recycling such as ultrafast endocytosis has added to the debate. Accumulating evidence further suggests that SV membrane retrieval and the reformation of functional SVs are separable processes. This may allow synapses to rapidly restore membrane surface area over a wide range of stimulations followed by slow reformation of release-competent SVs. One of the future challenges will be to pinpoint the exact mechanisms that link SV recycling modes to synaptic activity patterns at different synapses.

Dynamic Nuclear Polarization Provides New Insights into Chromophore Structure in Phytochrome Photoreceptors
Stöppler, D., Song(*), C., van Rossum, B. J., Geiger, M. A., Lang(*), C., Mroginski(*), M. A., Jagtap(*), A. P., Sigurdsson(*), S. T., Matysik(*), J., Hughes(*), J.; Oschkinat, H.
Angew Chem Int Ed Engl, 55:16017-16020
(2016)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Phytochromes are red/far-red photochromic photoreceptors acting as master regulators of development in higher plants, thereby controlling transcription of about 20 % of their genes. Light-induced isomerization of the bilin chromophore leads to large rearrangements in protein structure, whereby the role of protonation dynamics and charge distribution is of particular interest. To help unravel the inherent mechanisms, we present two-dimensional dynamic nuclear polarization (DNP) enhanced solid-state magic-angle spinning (MAS) NMR spectra of the functional sensory module of the cyanobacterial phytochrome Cph1. To this end, the pyrrole ring nitrogen signals were assigned unequivocally, enabling us to locate the positive charge of the phycocyanobilin (PCB) chromophore. To help analyze proton exchange pathways, the proximity of PCB ring nitrogen atoms and functionally relevant H2 O molecules was also determined. Our study demonstrates the value of DNP in biological solid-state MAS NMR spectroscopy.

Quantitative and Qualitative Analysis of Surface Modified Cellulose Utilizing TGA-MS
Loof(*), D., Hiller, M., Oschkinat, H.; Koschek(*), K.
Materials (Basel, Switzerland), 9
(2016)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: With the aim to enhance interfacial adhesion of a hydrophobic polymer matrix and cellulosic fibers and fillers, chemical surface modifications with silane coupling agents are performed. Thermogravimetric analysis (TGA) could be used to determine the degree of surface functionalization. However, similar thermal properties of treated and untreated cellulose hamper a precise determination of silane loading. This contribution deals with quantitative determination of silane loading combining both TGA and elemental analysis. Firstly, silane modified celluloses were studied by FT-IR, Raman, solid state NMR spectroscopy, and polarized light microscopy in order to determine functional groups and to study the impact of chemical treatment on cellulose morphology. Secondly, thermal stability and pyrolysis processes were studied by TG-MS analysis. In order to determine the exact silane loading, the mass percentages of the appropriate elements were quantified by elemental analysis and correlated with the charred residues determined by TGA yielding a linear dependency. With that correlation, it was possible to determine silane loadings for additional samples utilizing simple TGA measurements. The main advantage of that approach is that only one calibration is necessary for routine analyses of further samples and TGA-MS coupling gives additional information on thermal stability and pyrolysis routes, simultaneously.

Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides
Bertran-Vicente, J., Penkert, M., Nieto-Garcia, O., Jeckelmann(*), J. M., Schmieder, P., Krause, E.; Hackenberger, C. P.
Nat Commun, 7:12703
(2016)

Tags: Chemical Biology II (Hackenberger), Mass Spectrometry (Krause, E), Solution NMR (Schmieder)

Abstract: In contrast to protein O-phosphorylation, studying the function of the less frequent N- and S-phosphorylation events have lagged behind because they have chemical features that prevent their manipulation through standard synthetic and analytical methods. Here we report on the development of a chemoselective synthetic method to phosphorylate Cys side-chains in unprotected peptides. This approach makes use of a reaction between nucleophilic phosphites and electrophilic disulfides accessible by standard methods. We achieve the stereochemically defined phosphorylation of a Cys residue and verify the modification using electron-transfer higher-energy dissociation (EThcD) mass spectrometry. To demonstrate the use of the approach in resolving biological questions, we identify an endogenous Cys phosphorylation site in IICB(Glc), which is known to be involved in the carbohydrate uptake from the bacterial phosphotransferase system (PTS). This new chemical and analytical approach finally allows further investigating the functions and significance of Cys phosphorylation in a wide range of crucial cellular processes.

Stabilization of bacterially expressed erythropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylation sites
Hoffmann(*), E., Streichert, K., Nischan, N., Seitz(*), C., Brunner(*), T., Schwagerus, S., Hackenberger, C. P.; Rubini(*), M.
Mol Biosyst, 12:1750-1755
(2016)

Tags: Chemische Biologie II (Hackenberger)

Abstract: The covalent attachment of polyethylene glycol (PEG) to therapeutic proteins can improve their physicochemical properties. In this work we utilized the non-natural amino acid p-azidophenylalanine (pAzF) in combination with the chemoselective Staudinger-phosphite reaction to install branched PEG chains to recombinant unglycosylated erythropoietin (EPO) at each single naturally occurring glycosylation site. PEGylation with two short 750 or 2000 Da PEG units at positions 24, 38, or 83 significantly decreased unspecific aggregation and proteolytic degradation while biological activity in vitro was preserved or even increased in comparison to full-glycosylated EPO. This site-specific bioconjugation approach permits to analyse the impact of PEGylation at single positions. These results represent an important step towards the engineering of site-specifically modified EPO variants from bacterial expression with increased therapeutic efficacy.

Bis(arylmethyl)-substituted unsymmetrical phosphites for the synthesis of lipidated peptides via Staudinger-phosphite reactions
Nischan, N., Kasper, M. A., Mathew(*), T.; Hackenberger, C. P.
Org Biomol Chem, 14:7500-7508
(2016)

Tags: Chemical Biology II (Hackenberger)

Abstract: With this study we introduce new unsymmetrical phosphites to obtain lipidated peptide-conjugates starting from easily accessible azide-modified amino acid or peptide precursors. For this purpose, we investigated which substituents at alkyl phosphites lead to the highest formation of mono-alkylated phosphoramidate peptides. We found that phosphites containing one alkyl-chain and two picolyl or benzyl-substituents delivered alkyl phosphoramidate-conjugates in high yields, which also allowed a chemoselective lipidation of an unprotected azido polypeptide. Finally, monolipidated phosphoramidate peptides obtained by the unsymmetrical Staudinger phosphite reaction led to the formation of micelle-like structures and cellular uptake.

Current Status: Site-Specific Antibody Drug Conjugates
Schumacher, D., Hackenberger, C. P., Leonhardt(*), H.; Helma(*), J.
Journal of clinical immunology, 36 Suppl 1:100-107
(2016)

Tags: Chemical Biology II (Hackenberger)

Abstract: Antibody drug conjugates (ADCs), a promising class of cancer biopharmaceuticals, combine the specificity of therapeutic antibodies with the pharmacological potency of chemical, cytotoxic drugs. Ever since the first ADCs on the market, a plethora of novel ADC technologies has emerged, covering as diverse aspects as antibody engineering, chemical linker optimization and novel conjugation strategies, together aiming at constantly widening the therapeutic window for ADCs. This review primarily focuses on novel chemical and biotechnological strategies for the site-directed attachment of drugs that are currently validated for 2nd generation ADCs to promote conjugate homogeneity and overall stability.

Semi-synthesis of a tag-free O-GlcNAcylated tau protein by sequential chemoselective ligation
Schwagerus, S., Reimann, O., Despres(*), C., Smet-Nocca(*), C.; Hackenberger, C. P.
J Pept Sci, 22:327-333
(2016)

Tags: Chemical Biology II (Hackenberger)

Abstract: In this paper, the first semi-synthesis of the Alzheimer-relevant tau protein carrying an O-GlcNAcylation is demonstrated by using sequential chemoselective ligation. The 52-amino acid C-terminus of tau was obtained by native chemical ligation between two synthetic peptide fragments, one carrying the O-GlcNAc moiety on Ser400, which has recently been demonstrated to inhibit tau phosphorylation and to hinder tau oligomerization, and the other equipped with a photocleavable biotin handle. After desulfurization to deliver a native alanine at the ligation junction, the N-terminal cysteine was unmasked, and the peptide was further used for expressed protein ligation to generate the full-length tau protein, which was purified by a photocleavable biotin tag. We thus provide a synthetic route to obtain a homogenous tag-free O-GlcNAcylated tau protein that can further help to elucidate the significance of posttranslational modification on the tau protein and pave the way for evaluating possible drug targets in Alzheimer's disease. Copyright (c) 2016 European Peptide Society and John Wiley & Sons, Ltd.

Identification of Novel Nuclear Factor of Activated T Cell (NFAT)-associated Proteins in T Cells
Gabriel(*), C. H., Gross(*), F., Karl(*), M., Stephanowitz, H., Hennig(*), A. F., Weber(*), M., Gryzik(*), S., Bachmann(*), I., Hecklau(*), K., Wienands(*), J., Schuchhardt(*), J., Herzel(*), H., Radbruch(*), A., Krause, E.; Baumgrass(*), R.
J Biol Chem, 291:24172-24187
(2016)

Tags: Mass Spectrometry (Krause, E.)

Abstract: Transcription factors of the nuclear factor of activated T cell (NFAT) family are essential for antigen-specific T cell activation and differentiation. Their cooperative DNA binding with other transcription factors, such as AP1 proteins (FOS, JUN, and JUNB), FOXP3, IRFs, and EGR1, dictates the gene regulatory action of NFATs. To identify as yet unknown interaction partners of NFAT, we purified biotin-tagged NFATc1/alphaA, NFATc1/betaC, and NFATc2/C protein complexes and analyzed their components by stable isotope labeling by amino acids in cell culture-based mass spectrometry. We revealed more than 170 NFAT-associated proteins, half of which are involved in transcriptional regulation. Among them are many hitherto unknown interaction partners of NFATc1 and NFATc2 in T cells, such as Raptor, CHEK1, CREB1, RUNX1, SATB1, Ikaros, and Helios. The association of NFATc2 with several other transcription factors is DNA-dependent, indicating cooperative DNA binding. Moreover, our computational analysis discovered that binding motifs for RUNX and CREB1 are found preferentially in the direct vicinity of NFAT-binding motifs and in a distinct orientation to them. Furthermore, we provide evidence that mTOR and CHEK1 kinase activity influence NFAT's transcriptional potency. Finally, our dataset of NFAT-associated proteins provides a good basis to further study NFAT's diverse functions and how these are modulated due to the interplay of multiple interaction partners.

Inhibition of the key enzyme of sialic acid biosynthesis by C6-Se modified N-acetylmannosamine analogs
Nieto-Garcia, O., Wratil(*), P. R., Nguyen(*), L. D., Bohrsch(*), V., Hinderlich(*), S., Reutter(*), W.; Hackenberger, C. P. R.
Chem Sci, 7:3928-3933
(2016)

Tags: Chemical Biology II (Hackenberger)

Abstract: Synthetically accessible C6-analogs of N-acetylmannosamine (ManNAc) were tested as potential inhibitors of the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE/MNK), the key enzyme of sialic acid biosynthesis. Enzymatic experiments revealed that the modification introduced at the C6 saccharide position strongly influences the inhibitory potency. A C6-ManNAc diselenide dimer showed the strongest kinase inhibition in the low mu M range among all the substrates tested and successfully reduced cell surface sialylation in Jurkat cells.

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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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