FMP Publications

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

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References
A dataset comprising 141 magnetic resonance imaging scans of 98 extant sea urchin species
Ziegler(*), A., Faber(*), C., Mueller(*), S., Nagelmann(*), N.; Schröder, L.
GigaScience, 3:21
(2014)

Tags: Molecular Imaging (Schröder)

Abstract: BACKGROUND: Apart from its application in human diagnostics, magnetic resonance imaging (MRI) can also be used to study the internal anatomy of zoological specimens. As a non-invasive imaging technique, MRI has several advantages, such as rapid data acquisition, output of true three-dimensional imagery, and provision of digital data right from the onset of a study. Of particular importance for comparative zoological studies is the capacity of MRI to conduct high-throughput analyses of multiple specimens. In this study, MRI was applied to systematically document the internal anatomy of 98 representative species of sea urchins (Echinodermata: Echinoidea). FINDINGS: The dataset includes raw and derived image data from 141 MRI scans. Most of the whole sea urchin specimens analyzed were obtained from museum collections. The attained scan resolutions permit differentiation of various internal organs, including the digestive tract, reproductive system, coelomic compartments, and lantern musculature. All data deposited in the GigaDB repository can be accessed using open source software. Potential uses of the dataset include interactive exploration of sea urchin anatomy, morphometric and volumetric analyses of internal organs observed in their natural context, as well as correlation of hard and soft tissue structures. CONCLUSIONS: The dataset covers a broad taxonomical and morphological spectrum of the Echinoidea, focusing on 'regular' sea urchin taxa. The deposited files significantly expand the amount of morphological data on echinoids that are electronically available. The approach chosen here can be extended to various other vertebrate and invertebrate taxa. We argue that publicly available digital anatomical and morphological data gathered during experiments involving non-invasive imaging techniques constitute one of the prerequisites for future large-scale genotype-phenotype correlations.

Disturbed function of the blood-cerebrospinal fluid barrier aggravates neuro-inflammation
Kooij(*), G., Kopplin(*), K., Blasig, R., Stuiver(*), M., Koning(*), N., Goverse(*), G., van der Pol(*), S. M. A., Hof(*), B. V., Gollasch(*), M., Drexhage(*), J. A. R., Reijerkerk(*), A., Meij(*), I. C., Mebius(*), R., Willnow(*), T. E., Müller(*), D., Blasig, I. E.; de Vries(*), H. E.
Acta Neuropathol, 128:267-277
(2014)

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

Abstract: Multiple sclerosis (MS) is a chronic neuro-inflammatory disorder, which is marked by the invasion of the central nervous system by monocyte-derived macrophages and autoreactive T cells across the brain vasculature. Data from experimental animal models recently implied that the passage of leukocytes across the brain vasculature is preceded by their traversal across the blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus. The correlation between the presence of leukocytes in the CSF of patients suffering from MS and the number of inflammatory lesions as detected by magnetic resonance imaging suggests that inflammation at the choroid plexus contributes to the disease, although in a yet unknown fashion. We here provide first insights into the involvement of the choroid plexus in the onset and severity of the disease and in particular address the role of the tight junction protein claudin-3 (CLDN3) in this process. Detailed analysis of human post-mortem brain tissue revealed a selective loss of CLDN3 at the choroid plexus in MS patients compared to control tissues. Importantly, mice that lack CLDN3 have an impaired BCSFB and experience a more rapid onset and exacerbated clinical signs of experimental autoimmune encephalomyelitis, which coincides with enhanced levels of infiltrated leukocytes in their CSF. Together, this study highlights a profound role for the choroid plexus in the pathogenesis of multiple sclerosis, and implies that CLDN3 may be regarded as a crucial and novel determinant of BCSFB integrity.

Proteome analysis of the HIV-1 Gag interactome
Engeland(*), C. E., Brown(*), N. P., Borner(*), K., Schümann, M., Krause, E., Kaderali(*), L., Müller(*), G. A.; Kräusslich(*), H. G.
Virology, 460-461:194-206
(2014)

Tags: Mass Spectrometry (Krause, E.)

Abstract: Human immunodeficiency virus Gag drives assembly of virions in infected cells and interacts with host factors which facilitate or restrict viral replication. Although several Gag-binding proteins have been characterized, understanding of virus-host interactions remains incomplete. In a series of six affinity purification screens, we have identified protein candidates for interaction with HIV-1 Gag. Proteins previously found in virions or identified in siRNA screens for host factors influencing HIV-1 replication were recovered. Helicases, translation factors, cytoskeletal and motor proteins, factors involved in RNA degradation and RNA interference were enriched in the interaction data. Cellular networks of cytoskeleton, SR proteins and tRNA synthetases were identified. Most prominently, components of cytoplasmic RNA transport granules were co-purified with Gag. This study provides a survey of known Gag-host interactions and identifies novel Gag binding candidates. These factors are associated with distinct molecular functions and cellular pathways relevant in host-pathogen interactions.

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.

Clathrin terminal domain-ligand interactions regulate sorting of mannose 6-phosphate receptors mediated by AP-1 and GGA adaptors
Stahlschmidt, W., Robertson(*), M. J., Robinson(*), P. J., McCluskey(*), A.; Haucke, V.
J Biol Chem, 289:4906-4918
(2014)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Clathrin plays important roles in intracellular membrane traffic including endocytosis of plasma membrane proteins and receptors and protein sorting between the trans-Golgi network (TGN) and endosomes. Whether clathrin serves additional roles in receptor recycling, degradative sorting, or constitutive secretion has remained somewhat controversial. Here we have used acute pharmacological perturbation of clathrin terminal domain (TD) function to dissect the role of clathrin in intracellular membrane traffic. We report that internalization of major histocompatibility complex I (MHCI) is inhibited in cells depleted of clathrin or its major clathrin adaptor complex 2 (AP-2), a phenotype mimicked by application of Pitstop(R) inhibitors of clathrin TD function. Hence, MHCI endocytosis occurs via a clathrin/AP-2-dependent pathway. Acute perturbation of clathrin also impairs the dynamics of intracellular clathrin/adaptor complex 1 (AP-1)- or GGA (Golgi-localized, gamma-ear-containing, Arf-binding protein)-coated structures at the TGN/endosomal interface, resulting in the peripheral dispersion of mannose 6-phosphate receptors. By contrast, secretory traffic of vesicular stomatitis virus G protein, recycling of internalized transferrin from endosomes, or degradation of EGF receptor proceeds unperturbed in cells with impaired clathrin TD function. These data indicate that clathrin is required for the function of AP-1- and GGA-coated carriers at the TGN but may be dispensable for outward traffic en route to the plasma membrane.

Synthesis of the Pitstop family of clathrin inhibitors
Robertson(*), M. J., Deane(*), F. M., Stahlschmidt, W., von Kleist, L., Haucke, V., Robinson(*), P. J.; McCluskey(*), A.
Nat Protoc, 9:1592-1606
(2014)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: This protocol describes the synthesis of two classes of clathrin inhibitors, Pitstop 1 and Pitstop 2, along with two inactive analogs that can be used as negative controls (Pitstop inactive controls, Pitnot-2 and Pitnot-2-100). Pitstop-induced inhibition of clathrin TD function acutely interferes with clathrin-mediated endocytosis (CME), synaptic vesicle recycling and cellular entry of HIV, whereas clathrin-independent internalization pathways and secretory traffic proceed unperturbed; these reagents can, therefore, be used to investigate clathrin function, and they have potential pharmacological applications. Pitstop 1 is synthesized in two steps: sulfonation of 1,8-naphthalic anhydride and subsequent reaction with 4-amino(methyl)aniline. Pitnot-1 results from the reaction of 4-amino(methyl)aniline with commercially available 4-sulfo-1,8-naphthalic anhydride potassium salt. Reaction of 1-naphthalene sulfonyl chloride with pseudothiohydantoin followed by condensation with 4-bromobenzaldehyde yields Pitstop 2. The synthesis of the inactive control commences with the condensation of 4-bromobenzaldehyde with the rhodanine core. Thioketone methylation and displacement with 1-napthylamine affords the target compound. Although Pitstop 1-series compounds are not cell permeable, they can be used in biochemical assays or be introduced into cells via microinjection. The Pitstop 2-series compounds are cell permeable. The synthesis of these compounds does not require specialist equipment and can be completed in 3-4 d. Microwave irradiation can be used to reduce the synthesis time. The synthesis of the Pitstop 2 family is easily adaptable to enable the synthesis of related compounds such as Pitstop 2-100 and Pitnot-2-100. The procedures are also simple, efficient and amenable to scale-up, enabling cost-effective in-house synthesis for users of these inhibitor classes.

Semisynthesis and optimization of G protein-coupled receptor mimics
Abel, S., Geltinger, B., Heinrich, N., Michl, D., Klose, A., Beyermann, M.; Schwarzer(*), D.
J Pept Sci, 20:831-836
(2014)

Tags: Peptide Chemistry (Beyermann)

Abstract: We have recently developed a soluble mimic of the corticotropin-releasing factor receptor type 1 (CRF1), a membrane-spanning G protein-coupled receptor, which allowed investigations on receptor-ligand interactions. The CRF1 mimic consists of the receptor N-terminus and three synthetic extracellular loops (ECL1-3), which constitute the extracellular receptor domains (ECDs) of CRF1, coupled to a linear peptide template. Here, we report the synthesis of a modified CRF1 mimic, which is more similar to the native receptor possessing a cyclic template that displays the ECDs in a more physiological conformation compared with the initial linear design. In order to facilitate detailed biophysical investigations on CRF1 mimics, we have further established a cost-efficient access to the CRF1 mimic, which is suitable for isotopic labeling for NMR spectroscopy. To this end, the loop-mimicking cyclic peptide of the ECL2 of CRF1 was produced recombinantly and cyclized by expressed protein ligation. Cyclic ECL2 was obtained in milligram scale, and CRF1 mimics synthesized from this material displayed the same binding properties as synthetic CRF1 constructs.

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
(2014)

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
(2014)

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.

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
(2014)

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.

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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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