FMP Publications

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

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Physicochemical properties of cells and their effects on intrinsically disordered proteins (IDPs)
Theillet, F. X., Binolfi, A., Frembgen-Kesner(*), T., Hingorani(*), K., Sarkar(*), M., Kyne(*), C., Li(*), C., Crowley(*), P. B., Gierasch(*), L., Pielak(*), G. J., Elcock(*), A. H., Gershenson(*), A.; Selenko, P.
Chem Rev, 114:6661-6714

Tags: In-Cell NMR (Selenko)

Efficient modification of alpha-synuclein serine 129 by protein kinase CK1 requires phosphorylation of tyrosine 125 as a priming event
Kosten, J., Binolfi, A., Stuiver, M., Verzini, S., Theillet, F. X., Bekei, B., van Rossum, M.; Selenko, P.
Acs Chem Neurosci, 5:1203-1208

Tags: In-Cell NMR (Selenko)

Abstract: S129-phosphorylated alpha-synuclein (alpha-syn) is abundantly found in Lewy-body inclusions of Parkinson's disease patients. Residues neighboring S129 include the alpha-syn tyrosine phosphorylation sites Y125, Y133, and Y136. Here, we use time-resolved NMR spectroscopy to delineate atomic resolution insights into the modification behaviors of different serine and tyrosine kinases targeting these sites and show that Y125 phosphorylation constitutes a necessary priming event for the efficient modification of S129 by CK1, both in reconstituted kinase reactions and mammalian cell lysates. These results suggest that alpha-syn Y125 phosphorylation augments S129 modification under physiological in vivo conditions.

Non-stoichiometric O-acetylation of Shigella flexneri 2a O-specific polysaccharide: synthesis and antigenicity
Gauthier(*), C., Chassagne(*), P., Theillet, F. X., Guerreiro(*), C., Thouron(*), F., Nato(*), F., Delepierre(*), M., Sansonetti(*), P. J., Phalipon(*), A.; Mulard(*), L. A.
Organic & Biomolecular Chemistry, 12:4218-4232

Tags: In-Cell NMR (Selenko)

Abstract: Synthetic functional mimics of the O-antigen from Shigella flexneri 2a are seen as promising vaccine components against endemic shigellosis. Herein, the influence of the polysaccharide non-stoichiometric di-O-acetylation on antigenicity is addressed for the first time. Three decasaccharides, representing relevant internal mono-and di-O-acetylation profiles of the O-antigen, were synthesized from a pivotal protected decasaccharide designed to tailor late stage site-selective O-acetylation. The latter was obtained via a convergent route involving the imidate glycosylation chemistry. Binding studies to five protective mIgGs showed that none of the acetates adds significantly to broad antibody recognition. Yet, one of the five antibodies had a unique pattern of binding. With IC50 in the micromolar to submicromolar range mIgG F22-4 exemplifies a remarkable tight binding antibody against diversely O-acetylated and non-O-acetylated fragments of a neutral polysaccharide of medical importance.

Disorder and residual helicity alter p53-Mdm2 binding affinity and signaling in cells
Borcherds(*), W., Theillet, F. X., Katzer(*), A., Finzel(*), A., Mishall(*), K. M., Powell(*), A. T., Wu(*), H., Manieri(*), W., Dieterich(*), C., Selenko, P., Loewer(*), A.; Daughdrill(*), G. W.
Nat Chem Biol, 10:1000-1002

Tags: In-Cell NMR (Selenko)

Abstract: Levels of residual structure in disordered interaction domains determine in vitro binding affinities, but whether they exert similar roles in cells is not known. Here, we show that increasing residual p53 helicity results in stronger Mdm2 binding, altered p53 dynamics, impaired target gene expression and failure to induce cell cycle arrest upon DNA damage. These results establish that residual structure is an important determinant of signaling fidelity in cells.

Rapid proton-detected NMR assignment for proteins with fast magic angle spinning
Barbet-Massin(*), E., Pell(*), A. J., Retel, J. S., Andreas(*), L. B., Jaudzems(*), K., Franks, W. T., Nieuwkoop, A. J., Hiller, M., Higman(*), V., Guerry(*), P., Bertarello(*), A., Knight(*), M. J., Felletti(*), M., Le Marchand(*), T., Kotelovica(*), S., Akopjana(*), I., Tars(*), K., Stoppini(*), M., Bellotti(*), V., Bolognesi(*), M., Ricagno(*), S., Chou(*), J. J., Griffin(*), R. G., Oschkinat, H., Lesage(*), A., Emsley(*), L., Herrmann(*), T.; Pintacuda(*), G.
J Am Chem Soc, 136:12489-12497

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Using a set of six (1)H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5-30 kDa proteins. The approach relies on perdeuteration, amide (2)H/(1)H exchange, high magnetic fields, and high-spinning frequencies (omegar/2pi >/= 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary (13)C/(15)N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.

CLCN7 and TCIRG1 Mutations Differentially Affect Bone Matrix Mineralization in Osteopetrotic Individuals
Barvencik(*), F., Kurth(*), I., Koehne(*), T., Stauber, T., Zustin(*), J., Tsiakas, K., Ludwig, C. F., Beil(*), F. T., Pestka(*), J. M., Hahn(*), M., Santer(*), R., Supanchart(*), C., Kornak(*9, U., Del Fattore(*), A., Jentsch, T. J., Teti(*), A., Schulz(*), A., Schinke(*), T.; Amling(*), M.
J Bone Miner Res, 29:982-991

Tags: Physiology and Pathology of Ion Transport (Jentsch)

The cell surface proteome of Entamoeba histolytica
Biller(*), L., Matthiesen(*), J., Kühne(*), V., Lotter(*), H., Handal(*), G., Nozaki(*), T., Saito-Nakano(*), Y., Schümann, M., Roeder(*), T., Tannich(*), E., Krause, E.; Bruchhaus(*), I.
Mol Cell Proteomics, 13:132-144

Tags: Mass Spectrometry (Krause, E.)

Abstract: Surface molecules are of major importance for host-parasite interactions. During Entamoeba histolytica infections, these interactions are predicted to be of prime importance for tissue invasion, induction of colitis and liver abscess formation. To date, however, little is known about the molecules involved in these processes, with only about 20 proteins or protein families found exposed on the E. histolytica surface. We have therefore analyzed the complete surface proteome of E. histolytica. Using cell surface biotinylation and mass spectrometry, 693 putative surface-associated proteins were identified. In silico analysis predicted that approximately 26% of these proteins are membrane-associated, as they contain transmembrane domains and/or signal sequences, as well as sites of palmitoylation, myristoylation, or prenylation. An additional 25% of the identified proteins likely represent nonclassical secreted proteins. Surprisingly, no membrane-association sites could be predicted for the remaining 49% of the identified proteins. To verify surface localization, 23 proteins were randomly selected and analyzed by immunofluorescence microscopy. Of these 23 proteins, 20 (87%) showed definite surface localization. These findings indicate that a far greater number of E. histolytica proteins than previously supposed are surface-associated, a phenomenon that may be based on the high membrane turnover of E. histolytica.

N-[6-(4-butanoyl-5-methyl-1H-pyrazol-1-yl)pyridazin-3-yl]-5-chloro-1-[2-(4-methyl piperazin-1-yl)-2-oxoethyl]-1H-indole-3-carboxamide (SAR216471), a novel intravenous and oral, reversible, and directly acting P2Y12 antagonist
Boldron(*), C., Besse(*), A., Bordes(*), M. F., Tissandie(*), S., Yvon(*), X., Gau(*), B., Badorc(*), A., Rousseaux(*), T., Barre(*), G., Meneyrol(*), J., Zech(*), G., Nazare, M., Fossey(*), V., Pflieger(*), A. M., Bonnet-Lignon(*), S., Millet(*), L., Briot(*), C., Dol(*), F., Herault(*), J. P., Savi(*), P., Lassalle(*), G., Delesque(*), N., Herbert(*), J. M.; Bono(*), F.
Journal of medicinal chemistry, 57:7293-7316

Tags: Medicinal Chemistry (Nazare)

Abstract: In the search of a potential backup for clopidogrel, we have initiated a HTS campaign designed to identify novel reversible P2Y12 antagonists. Starting from a hit with low micromolar binding activity, we report here the main steps of the optimization process leading to the identification of the preclinical candidate SAR216471. It is a potent, highly selective, and reversible P2Y12 receptor antagonist and by far the most potent inhibitor of ADP-induced platelet aggregation among the P2Y12 antagonists described in the literature. SAR216471 displays potent in vivo antiplatelet and antithrombotic activities and has the potential to differentiate from other antiplatelet agents.

Multivalent presentation of the cell-penetrating peptide nona-arginine on a linear scaffold strongly increases its membrane-perturbing capacity
Chakrabarti(*), A., Witsenburg(*), J. J., Sinzinger(*), M. D., Richter, M., Wallbrecher(*), R., Cluitmans(*), J. C., Verdurmen(*), W. P., Tanis(*), S., Adjobo-Hermans(*), M. J., Rademann, J.; Brock(*), R.
Biochim Biophys Acta, 1838:3097-3106

Tags: Medicinal Chemistry (Rademann)

Abstract: Arginine-rich cell-penetrating peptides (CPP) are widely employed as delivery vehicles for a large variety of macromolecular cargos. As a mechanism-of-action for induction of uptake cross-linking of heparan sulfates and interaction with lipid head groups have been proposed. Here, we employed a multivalent display of the CPP nona-arginine (R9) on a linear dextran scaffold to assess the impact of heparan sulfate and lipid interactions on uptake and membrane perturbation. Increased avidity through multivalency should potentiate molecular phenomena that may only play a minor role if only individual peptides are used. To this point, the impact of multivalency has only been explored for dendrimers, CPP-decorated proteins and nanoparticles. We reasoned that multivalency on a linear scaffold would more faithfully mimic the arrangement of peptides at the membrane at high local peptide concentrations. On average, five R9 were coupled to a linear dextran backbone. The conjugate displayed a direct cytoplasmic uptake similar to free R9 at concentrations higher than 10muM. However, this uptake was accompanied by an increased membrane disturbance and cellular toxicity that was independent of the presence of heparan sulfates. In contrast, for erythrocytes, the multivalent conjugate induced aggregation, however, showed only limited membrane perturbation. Overall, the results demonstrate that multivalency of R9 on a linear scaffold strongly increases the capacity to interact with the plasma membrane. However, the induction of membrane perturbation is a function of the cellular response to peptide binding.

Reporter assay for endo/lysosomal escape of toxin-based therapeutics
Gilabert-Oriol(*), R., Thakur(*), M., von Mallinckrodt(*), B., Bhargava(*), C., Wiesner, B., Eichhorst, J., Melzig(*), M. F., Fuchs(*), H.; Weng(*), A.
Toxins (Basel), 6:1644-1666

Tags: Cellular Imaging (Wiesner)

Abstract: Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters-horseradish peroxidase (HRP), Alexa Fluor 488 (Alexa) and ricin A-chain (RTA)-were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates-saporin-HRP, (Alexa)saporin and saporin-KQ-RTA-were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of (Alexa)saporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10-1000 nM.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)

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