FMP Publications

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

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References
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
(2014)

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.

Development of 1,8-naphthalimides as clathrin inhibitors
MacGregor(*), K. A., Robertson(*), M. J., Young(*), K. A., von Kleist(*), L., Stahlschmidt, W., Whiting(*), A., Chau(*), N., Robinson(*), P. J., Haucke, V.; McCluskey(*), A.
Journal of medicinal chemistry, 57:131-143
(2014)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: We reported the first small molecule inhibitors of the interaction between the clathrin N-terminal domain (TD) and endocyctic accessory proteins (i.e., clathrin inhibition1). Initial screening of a approximately 17 000 small molecule ChemBioNet library identified 1. Screening of an existing in-house propriety library identified four substituted 1,8-napthalimides as approximately 80-120 muM clathrin inhibitors. Focused library development gave 3-sulfo-N-(4-aminobenzyl)-1,8-naphthalimide, potassium salt (18, IC50 approximately 18 muM). A second library targeting the 4-aminobenzyl moiety was developed, and four analogues displayed comparable activity (26, 27, 28, 34 with IC50 values of 22, 16, 15, and 15 muM respectively) with a further four (24, 25, 32, 33) more active than 18 with IC50 values of 10, 6.9, 12, and 10 muM, respectively. Docking studies rationalized the structure-activity relationship (SAR) with the biological data. 3-Sulfo-N-benzyl-1,8-naphthalimide, potassium salt (25) with an IC50 approximately 6.9 muM, is the most potent clathrin terminal domain-amphiphysin inhibitor reported to date.

Activation of Ligand Binding Domains of an AMPA-Type Glutamate Receptor
Baranovic, J., Chebli, M., Salazar, H. P., Faelber(*), K., Ghisi, V., Lau(*), A. Y., Daumke(*), O.; Plested, A. J. R.
Biophys. J., 106:29a-29a
(2014)

Tags: Molecular Neuroscience and Biophysics (Plested)

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
(2014)

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.

Molecular characterization of ubiquitin-specific protease 18 reveals substrate specificity for interferon-stimulated gene 15
Basters(*), A., Geurink(*), P. P., El Oualid(*), F., Ketscher(*), L., Casutt(*), M. S., Krause, E., Ovaa(*), H., Knobeloch(*), K. P.; Fritz(*), G.
Febs J, 281:1918-1928
(2014)

Tags: Mass Spectrometry (Krause, E.)

Abstract: UNLABELLED: Protein modification by interferon-stimulated gene 15 (ISG15), an ubiquitin-like modifier, affects multiple cellular functions and represents one of the major antiviral effector systems. Covalent linkage of ISG15 to proteins was previously reported to be counteracted by ubiquitin-specific protease 18 (USP18). To date, analysis of the molecular properties of USP18 was hampered by low expression yields and impaired solubility. We established high-yield expression of USP18 in insect cells and purified the protease to homogeneity. USP18 binds with high affinity to ISG15, as shown by microscale thermophoresis with a Kd of 1.3 +/- 0.2 mum. The catalytic properties of USP18 were characterized by a novel assay using ISG15 fused to a fluorophore via an isopeptide bond, giving a Km of 4.6 +/- 0.2 mum and a kcat of 0.23 +/- 0.004 s(-1) , respectively, at pH 7.5. Furthermore, the recombinant enzyme cleaves efficiently ISG15 but not ubiquitin from endogenous cellular substrates. In line with these data, USP18 exhibited neither cross-reactivity with an ubiquitin isopeptide fluorophore substrate, nor with a ubiquitin vinyl sulfone, showing that the enzyme is specific for ISG15. STRUCTURED DIGITAL ABSTRACT: ISG15 and USP18 bind by microscale thermophoresis (View interaction) USP18 cleaves ISG15 by enzymatic study (View interaction).

Mechanism of Modulation of AMPA Receptors by Stargazin
Carbone, A. L.; Plested, A. J.
Biophys. J., 106:150a-150a
(2014)

Tags: Molecular Neuroscience and Biophysics (Plested)

PKA phosphorylation of p62/SQSTM1 regulates PB1 domain interaction partner binding
Christian(*), F., Krause, E., Houslay(*), M. D.; Baillie(*), G. S.
Bba-Mol Cell Res, 1843:2765-2774
(2014)

Tags: Mass Spectrometry (Krause, E.)

Abstract: p62, also known as SQSTM1, is a multi-domain signalling scaffold protein involved in numerous critical cellular functions such as autophagy, apoptosis and inflammation. Crucial interactions relevant to these functions are mediated by the N-terminal Phox and Bem1p (PB1) domain, which is divided into two interaction surfaces, one of predominantly acidic and one of basic character. Most known interaction partners, including atypical protein kinase C (aPKC), bind to the basic surface, and acidic-basic interactions at this interface also allow for p62 homopolymerisation. We identify here that the coupling of p62 to the cAMP signalling system is conferred by both the direct binding of cAMP degrading phosphodiesterase-4 (PDE4) to the acidic surface of the p62 PB1 domain and the phosphorylation of the basic surface of this domain by CAMP-dependent protein kinase (PKA). Such phosphoiylation is a previously unknown means of regulating PB1 domain interaction partnerships by disrupting the interaction of p62 with basic surface binding partners, such as aPKCs, as well as p62 homopolymerisation. Thus, we uncover a new regulatory mechanism that connects cAMP signalling with the p62 multi-domain signalling scaffold and autophagy cargo receptor protein. (C) 2014 Elsevier B.V. All rights reserved.

PKA phosphorylation of p62/SQSTM1 regulates PB1 domain interaction partner binding
Christian(*), F., Krause, E., Houslay(*), M. D.; Baillie(*), G. S.
Biochim Biophys Acta, 1843:2765-2774
(2014)

Tags: Mass Spectrometry (Krause, E.)

Abstract: p62, also known as SQSTM1, is a multi-domain signalling scaffold protein involved in numerous critical cellular functions such as autophagy, apoptosis and inflammation. Crucial interactions relevant to these functions are mediated by the N-terminal Phox and Bem1p (PB1) domain, which is divided into two interaction surfaces, one of predominantly acidic and one of basic character. Most known interaction partners, including atypical protein kinase C (aPKC), bind to the basic surface, and acidic-basic interactions at this interface also allow for p62 homopolymerisation. We identify here that the coupling of p62 to the cAMP signalling system is conferred by both the direct binding of cAMP degrading phosphodiesterase-4 (PDE4) to the acidic surface of the p62 PB1 domain and the phosphorylation of the basic surface of this domain by cAMP-dependent protein kinase (PKA). Such phosphorylation is a previously unknown means of regulating PB1 domain interaction partnerships by disrupting the interaction of p62 with basic surface binding partners, such as aPKCs, as well as p62 homopolymerisation. Thus, we uncover a new regulatory mechanism that connects cAMP signalling with the p62 multi-domain signalling scaffold and autophagy cargo receptor protein.

PI3K class II alpha controls spatially restricted endosomal PtdIns3P and Rab11 activation to promote primary cilium function
Franco(*), I., Gulluni(*), F., Campa(*), C. C., Costa(*), C., Margaria(*), J. P., Ciraolo(*), E., Martini(*), M., Monteyne(*), D., De Luca(*), E., Germena(*), G., Posor, Y., Maffucci(*), T., Marengo(*), S., Haucke, V., Falasca(*), M., Perez-Morga(*), D., Boletta(*), A., Merlo(*), G. R.; Hirsch(*), E.
Dev Cell, 28:647-658
(2014)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Multiple phosphatidylinositol (PtdIns) 3-kinases (PI3Ks) can produce PtdIns3P to control endocytic trafficking, but whether enzyme specialization occurs in defined subcellular locations is unclear. Here, we report that PI3K-C2alpha is enriched in the pericentriolar recycling endocytic compartment (PRE) at the base of the primary cilium, where it regulates production of a specific pool of PtdIns3P. Loss of PI3K-C2alpha-derived PtdIns3P leads to mislocalization of PRE markers such as TfR and Rab11, reduces Rab11 activation, and blocks accumulation of Rab8 at the primary cilium. These changes in turn cause defects in primary cilium elongation, Smo ciliary translocation, and Sonic Hedgehog (Shh) signaling and ultimately impair embryonic development. Selective reconstitution of PtdIns3P levels in cells lacking PI3K-C2alpha rescues Rab11 activation, primary cilium length, and Shh pathway induction. Thus, PI3K-C2alpha regulates the formation of a PtdIns3P pool at the PRE required for Rab11 and Shh pathway activation.

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
(2014)

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.

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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 
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info(at)fmp-berlin.de

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