FMP Publications

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

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References
High-resolution structure of the Shigella type-III secretion needle by solid-state NMR and cryo-electron microscopy
Demers, J. P., Habenstein(*), B., Loquet(*), A., Kumar Vasa(*), S., Giller(*), K., Becker(*), S., Baker, D., Lange, A.; Sgourakis(*), N. G.
Nat Commun, 5:4976
(2014)

Tags: Molecular Biophysics (Lange, A.)

Abstract: We introduce a general hybrid approach for determining the structures of supramolecular assemblies. Cryo-electron microscopy (cryo-EM) data define the overall envelope of the assembly and rigid-body orientation of the subunits while solid-state nuclear magnetic resonance (ssNMR) chemical shifts and distance constraints define the local secondary structure, protein fold and inter-subunit interactions. Finally, Rosetta structure calculations provide a general framework to integrate the different sources of structural information. Combining a 7.7-A cryo-EM density map and 996 ssNMR distance constraints, the structure of the type-III secretion system needle of Shigella flexneri is determined to a precision of 0.4 A. The calculated structures are cross-validated using an independent data set of 691 ssNMR constraints and scanning transmission electron microscopy measurements. The hybrid model resolves the conformation of the non-conserved N terminus, which occupies a protrusion in the cryo-EM density, and reveals conserved pore residues forming a continuous pattern of electrostatic interactions, thereby suggesting a mechanism for effector protein translocation.

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.

Disrupting MLC1 and GlialCAM and ClC-2 interactions in leukodystrophy entails glial chloride channel dysfunction
Hoegg-Beiler, M. B., Sirisi(*), S., Orozco, I. J., Ferrer(*), I., Hohensee, S., Auberson, M., Gödde, K., Vilches(*), C., de Heredia(*), M. L., Nunes(*), V., Estevez(*), R.; Jentsch, T. J.
Nat Commun, 5:3475
(2014)

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Defects in the astrocytic membrane protein MLC1, the adhesion molecule GlialCAM or the chloride channel ClC-2 underlie human leukoencephalopathies. Whereas GlialCAM binds ClC-2 and MLC1, and modifies ClC-2 currents in vitro, no functional connections between MLC1 and ClC-2 are known. Here we investigate this by generating loss-of-function Glialcam and Mlc1 mouse models manifesting myelin vacuolization. We find that ClC-2 is unnecessary for MLC1 and GlialCAM localization in brain, whereas GlialCAM is important for targeting MLC1 and ClC-2 to specialized glial domains in vivo and for modifying ClC-2's biophysical properties specifically in oligodendrocytes (OLs), the cells chiefly affected by vacuolization. Unexpectedly, MLC1 is crucial for proper localization of GlialCAM and ClC-2, and for changing ClC-2 currents. Our data unmask an unforeseen functional relationship between MLC1 and ClC-2 in vivo, which is probably mediated by GlialCAM, and suggest that ClC-2 participates in the pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts.

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.

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.

Cerebral blood volume estimation by ferumoxytol-enhanced steady-state MRI at 9.4 T reveals microvascular impact of alpha1 -adrenergic receptor antibodies
Pohlmann(*), A., Karczewski(*), P., Ku(*), M. C., Dieringer(*), B., Waiczies(*), H., Wisbrun, N., Kox(*), S., Palatnik(*), I., Reimann(*), H. M., Eichhorn(*), C., Waiczies(*), S., Hempel(*), P., Lemke(*), B., Niendorf(*), T.; Bimmler(*), M.
Nmr Biomed, 27:1085-1093
(2014)

Tags: Animal Facility (Wiesbrun)

Abstract: Cerebrovascular abnormality is frequently accompanied by cognitive dysfunctions, such as dementia. Antibodies against the alpha1 -adrenoceptor (alpha1 -AR) can be found in patients with Alzheimer's disease with cerebrovascular disease, and have been shown to affect the larger vessels of the brain in rodents. However, the impact of alpha1 -AR antibodies on the cerebral vasculature remains unclear. In the present study, we established a neuroimaging method to measure the relative cerebral blood volume (rCBV) in small rodents with the ultimate goal to detect changes in blood vessel density and/or vessel size induced by alpha1 -AR antibodies. For this purpose, mapping of R2 * and R2 was performed using MRI at 9.4 T, before and after the injection of intravascular iron oxide particles (ferumoxytol). The change in the transverse relaxation rates (DeltaR2 *, DeltaR2 ) showed a significant rCBV decrease in the cerebrum, cortex and hippocampus of rats (except hippocampal DeltaR2 ), which was more pronounced for DeltaR2 * than for DeltaR2 . Immunohistological analyses confirmed that the alpha1 -AR antibody induced blood vessel deficiencies. Our findings support the hypothesis that alpha1 -AR antibodies lead to cerebral vessel damage throughout the brain, which can be monitored by MRI-derived rCBV, a non-invasive neuroimaging method. This demonstrates the value of rCBV estimation by ferumoxytol-enhanced MRI at 9.4 T, and further underlines the significance of this antibody in brain diseases involving vasculature impairments, such as dementia.

Stereoselective Synthesis of Proline- Derived Dipeptide Scaffolds ( ProM-3 and ProM-7) Rigidified in a PPII Helix Conformation
Reuter(*), C., Kleczka(*), M., de Mazancourt(*), S., Neudörfl(*), J. M., Kühne, R.; Schmalz(*), H. G.
Eur J Org Chem, 2014:2664-2667
(2014)

Tags: Computational Chemistry and Protein Design (Kühne)

Abstract: Following a peptide coupling/metathesis-based strategy, the two diastereomeric scaffolds ProM-3 and ProM-7 were stereoselectively synthesized (as 9-fluorenylmethoxycarbonyl derivatives), and their configuration was unambiguously proven by means of X-ray crystallography. The required dehydroisoleucine building blocks were prepared by applying the enantioselective Kazmaier-Claisen rearrangement. The target compounds represent dipeptide analogs rigidified in a PPII helix conformation, which are of interest for the development of new proteomimetics that selectively bind to protein domains specialized in the recognition of ligands adopting a PPII helix secondary structure.

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.

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