FMP Publications

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

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References
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
(2014)

Tags: Physiology and Pathology of Ion Transport (Jentsch)

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.

Ultrafast CEST imaging
Döpfert, J., Zaiss(*), M., Witte, C.; Schröder, L.
J Magn Reson, 243:47-53
(2014)

Tags: Molecular Imaging (Schröder)

Abstract: We describe a new MR imaging method for the rapid characterization or screening of chemical exchange saturation transfer (CEST) contrast agents. It is based on encoding the chemical shift dimension with an additional gradient as proposed in previous ultrafast CEST spectroscopy approaches, but extends these with imaging capabilities. This allows us to investigate multiple compounds simultaneously with an arbitrary sample tube arrangement. The technique requires a fast multislice readout to ensure the saturation is not lost during data acquisition due to T1 relaxation. We therefore employ radial subsampling, acquiring only 10 projections per CEST image with a 128x128 matrix. To recover the images, we use a heuristic reconstruction algorithm that incorporates low rank and limited object support as prior knowledge. This way, we are able to acquire a spectral CEST data set consisting of 15 saturation offsets more than 16 times faster than compared with conventional CEST imaging.

Site-Specific Copper-Catalyzed Oxidation of alpha-Synuclein: Tightening the Link between Metal Binding and Protein Oxidative Damage in Parkinson's Disease
Miotto(*), M. C., Rodriguez(*), E. E., Valiente-Gabioud(*), A. A., Torres-Monserrat(*), V., Binolfi, A., Quintanar(*), L., Zweckstetter(*), M., Griesinger(*), C.; Fernandez(*), C. O.
Inorg Chem, 53:4350-4358
(2014)

Tags: In-Cell NMR (Selenko)

Abstract: Amyloid aggregation of a-synuclein (AS) has been linked to the pathological effects associated with Parkinson's disease (PD). Cu-II binds specifically at the N-terminus of AS and triggers its aggregation. Site-specific Cu-I-catalyzed oxidation of AS has been proposed as a plausible mechanism for metal-enhanced AS amyloid formation. In this study, Cu-I binding to AS was probed by NMR spectroscopy, in combination with synthetic peptide models, site-directed mutagenesis, and C-terminal-truncated protein variants. Our results demonstrate that both Met residues in the motif (MDVFM5)-M-1 constitute key structural determinants for the high-affinity binding of Cu-I to the N-terminal region of AS. The replacement of one Met residue by Ile causes a dramatic decrease in the binding affinity for Cu-I, whereas the removal of both Met residues results in a complete lack of binding. Moreover, these Met residues can be oxidized rapidly after air exposure of the AS-Cu-I complex, whereas Met-116 and Met-127 in the C-terminal region remain unaffected. Met-1 displays higher susceptibility to oxidative damage compared to Met-5 because it is directly involved in both Cu-II and Cu-I coordination, resulting in closer exposure to the reactive oxygen species that may be generated by the redox cycling of copper. Our findings support a mechanism where the interaction of AS with copper ions leads to site-specific metal-catalyzed oxidation in the protein under physiologically relevant conditions. In light of recent biological findings, these results support a role for AS-copper interactions in neurodegeneration in PD.

Bioinorganic chemistry of synucleinopathies: deciphering the binding features of Met motifs and His-50 in AS-Cu(I) interactions
Miotto(*), M. C., Binolfi, A., Zweckstetter(*), M., Griesinger(*), C.; Fernandez(*), C. O.
J Inorg Biochem, 141:208-211
(2014)

Tags: In-Cell NMR (Selenko)

Abstract: The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinson's disease (PD) and other neurodegenerative synucleinopathies. This process is selectively enhanced by copper in vitro and the interaction is proposed to play a potential role in vivo. Presently, the identity of the Cu(I) binding sites in AS and their relative affinities are under debate. In this work we have addressed unresolved details related to the structural binding specificity and affinity of Cu(I) to full-length AS. We demonstrated conclusively that: (i) the binding preferences of Cu(I) for the Met-binding sites at the N- (Kd=20 muM) and C-terminus (Kd=270 muM) of AS are widely different: (ii) the imidazole ring of His-50 acts as an effective anchoring residue (Kd=50 muM) for Cu(I) binding to AS; and (iii) no major structural rearrangements occur in the protein upon Cu(I) binding. Overall, our work shows that Cu(I) binding to the N- and C-terminal regions of AS are two independent events, with substantial differences in their affinities, and suggest that protein oxidative damage derived from a misbalance in cellular copper homeostasis would target preferentially the N-terminal region of AS. This knowledge is key to understanding the structural-aggregation basis of the copper catalyzed oxidation of AS.

Interferon-gamma safeguards blood-brain barrier during experimental autoimmune encephalomyelitis
Ni(*), C., Wang(*), C., Zhang(*), J., Qu(*), L., Liu(*), X., Lu(*), Y., Yang(*), W., Deng(*), J., Lorenz, D., Gao(*), P., Meng(*), Q., Yan(*), X., Blasig, I. E.; Qin(*), Z.
The American journal of pathology, 184:3308-3320
(2014)

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

Abstract: The function of blood-brain barrier is often disrupted during the progression of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the molecular mechanism of blood-brain barrier modulation during neuroinflammation remains unclear. Herein, we show that the expression of interferon-gamma (IFNgamma) receptor on endothelial cells (ECs) protected mice from the brain inflammation during EAE. IFNgamma stabilized the integrity of the cerebral endothelium and prevented the infiltration of leukocytes into the brain. Further analysis revealed that IFNgamma increased the expression of tight junction proteins zonula occludens protein 1 and occludin, as well as membranous distribution of claudin-5, in brain ECs. Silencing claudin-5 abolished the IFNgamma-mediated improvement of EC integrity. Taken together, our results show that IFNgamma, a pleiotropic proinflammatory cytokine, stabilizes blood-brain barrier integrity and, therefore, prevents brain inflammation during EAE.

A presynaptic role for the cytomatrix protein GIT in synaptic vesicle recycling
Podufall, J., Tian(*), R., Knoche(*), E., Puchkov, D., Walter, A. M., Rosa(*), S., Quentin(*), C., Vukoja, A., Jung(*), N., Lampe, A., Wichmann(*), C., Böhme(*), M., Depne(*)r, H., Zhang(*), Y. Q., Schmoranzer, J., Sigrist(*), S. J.; Haucke, V.
Cell Rep, 7:1417-1425
(2014)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Neurotransmission involves the exo-endocytic cycling of synaptic vesicles (SVs) within nerve terminals. Exocytosis is facilitated by a cytomatrix assembled at the active zone (AZ). The precise spatial and functional relationship between exocytic fusion of SVs at AZ membranes and endocytic SV retrieval is unknown. Here, we identify the scaffold G protein coupled receptor kinase 2 interacting (GIT) protein as a component of the AZ-associated cytomatrix and as a regulator of SV endocytosis. GIT1 and its D. melanogaster ortholog, dGIT, are shown to directly associate with the endocytic adaptor stonin 2/stoned B. In Drosophila dgit mutants, stoned B and synaptotagmin levels are reduced and stoned B is partially mislocalized. Moreover, dgit mutants show morphological and functional defects in SV recycling. These data establish a presynaptic role for GIT in SV recycling and suggest a connection between the AZ cytomatrix and the endocytic machinery.

A missense mutation accelerating the gating of the lysosomal Cl-/H+-exchanger ClC-7/Ostm1 causes osteopetrosis with gingival hamartomas in cattle
Sartelet(*), A., Stauber, T., Coppieters(*), W., Ludwig, C. F., Fasquelle(*), C., Druet(*), T., Zhang(*), Z. Y., Ahariz(*), N., Cambisano(*), N., Jentsch, T. J.; Charlier(*), C.
Dis Model Mech, 7:119-128
(2014)

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Chloride-proton exchange by the lysosomal anion transporter ClC7/Ostm1 is of pivotal importance for the physiology of lysosomes and bone resorption. Mice lacking either ClC-7 or Ostm1 develop a lysosomal storage disease and mutations in either protein have been found to underlie osteopetrosis in mice and humans. Some human disease-causing CLCN7 mutations accelerate the usually slow voltage-dependent gating of ClC-7/Ostm1. However, it has remained unclear whether the fastened kinetics is indeed causative for the disease. Here we identified and characterized a new deleterious ClC-7 mutation in Belgian Blue cattle with a severe symptomatology including perinatal lethality and in most cases gingival hamartomas. By autozygosity mapping and genome-wide sequencing we found a handful of candidate variants, including a cluster of three private SNPs causing the substitution of a conserved tyrosine in the CBS2 domain of ClC-7 by glutamine. The case for ClC-7 was strengthened by subsequent examination of affected calves that revealed severe osteopetrosis. The Y750Q mutation largely preserved the lysosomal localization and assembly of ClC-7/Ostm1, but drastically accelerated its activation by membrane depolarization. These data provide first evidence that accelerated ClC-7/Ostm1 gating per se is deleterious, highlighting a physiological importance of the slow voltage-activation of ClC-7/Ostm1 in lysosomal function and bone resorption.

Liposome reconstitution and modulation of recombinant prenylated human Rac1 by GEFs, GDI1 and Pak1
Zhang(*), S. C., Gremer(*), L., Heise(*), H., Janning(*), P., Shymanets(*), A., Cirstea(*), I. C., Krause, E., Nürnberg(*), B.; Ahmadian(*), M. R.
Plos One, 9:e102425
(2014)

Tags: Mass Spectrometry (Krause, E.)

Abstract: Small Rho GTPases are well known to regulate a variety of cellular processes by acting as molecular switches. The regulatory function of Rho GTPases is critically dependent on their posttranslational modification at the carboxyl terminus by isoprenylation and association with proper cellular membranes. Despite numerous studies, the mechanisms of recycling and functional integration of Rho GTPases at the biological membranes are largely unclear. In this study, prenylated human Rac1, a prominent member of the Rho family, was purified in large amount from baculovirus-infected Spodoptera frugiperda insect cells using a systematic detergent screening. In contrast to non-prenylated human Rac1 purified from Escherichia coli, prenylated Rac1 from insect cells was able to associate with synthetic liposomes and to bind Rho-specific guanine nucleotide dissociation inhibitor 1 (GDI1). Subsequent liposome reconstitution experiments revealed that GDI1 efficiently extracts Rac1 from liposomes preferentially in the inactive GDP-bound state. The extraction was prevented when Rac1 was activated to its GTP-bound state by Rac-specific guanine nucleotide exchange factors (GEFs), such as Vav2, Dbl, Tiam1, P-Rex1 and TrioN, and bound by the downstream effector Pak1. We found that dissociation of Rac1-GDP from its complex with GDI1 strongly correlated with two distinct activities of especially Dbl and Tiam1, including liposome association and the GDP/GTP exchange. Taken together, our results provided first detailed insights into the advantages of the in vitro liposome-based reconstitution system to study both the integration of the signal transducing protein complexes and the mechanisms of regulation and signaling of small GTPases at biological membranes.

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.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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