FMP Publications

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

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References
Modulation of monocarboxylate transporter 8 oligomerization by specific pathogenic mutations
Fischer(*), J., Kleinau(*), G., Müller(*), A., Kühnen(*), P., Zwanziger(*), D., Kinne, A., Rehders(*), M., Moeller(*), L. C., Führer(*), D., Grüters(*), A., Krude(*), H., Brix(*), K.; Biebermann(*), H.
Journal of molecular endocrinology, 54:39-50
(2015)

Tags: Structural Bioinformatics and Protein Design (Krause, G.)

Abstract: The monocarboxylate transporter 8 (MCT8) is a member of the major facilitator superfamily (MFS). These membrane-spanning proteins facilitate translocation of a variety of substrates, MCT8 specifically transports iodothyronines. Mutations in MCT8 are the underlying cause of severe X-linked psychomotor retardation. At the molecular level, such mutations led to deficiencies in substrate translocation due to reduced cell-surface expression, impaired substrate binding, or decreased substrate translocation capabilities. However, the causal relationships between genotypes, molecular features of mutated MCT8, and patient characteristics have not yet been comprehensively deciphered. We investigated the relationship between pathogenic mutants of MCT8 and their capacity to form dimers (presumably oligomeric structures) as a potential regulatory parameter of the transport function of MCT8. Fourteen pathogenic variants of MCT8 were investigated in vitro with respect to their capacity to form oligomers. Particular mutations close to the substrate translocation channel (S194F, A224T, L434W, and R445C) were found to inhibit dimerization of MCT8. This finding is in contrast to those for other transporters or transmembrane proteins, in which substitutions predominantly at the outer-surface inhibit oligomerization. Moreover, specific mutations of MCT8 located in transmembrane helix 2 (del230F, V235M, and ins236V) increased the capacity of MCT8 variants to dimerize. We analyzed the localization of MCT8 dimers in a cellular context, demonstrating differences in MCT8 dimer formation and distribution. In summary, our results add a new link between the functions (substrate transport) and protein organization (dimerization) of MCT8, and might be of relevance for other members of the MFS. Finally, the findings are discussed in relationship to functional data combined with structural-mechanistical insights into MCT8.

Strategies for solid-state NMR investigations of supramolecular assemblies with large subunit sizes
Fricke, P., Chevelkov, V., Shi, C.; Lange, A.
J Magn Reson, 253:2-9
(2015)

Tags: Molecular Biophysics (Lange, A.)

Abstract: Solid-state NMR is a versatile tool to study structure and dynamics of insoluble and non-crystalline biopolymers. Supramolecular protein assemblies are formed by self-association of multiple copies of single small-sized proteins. Because of their high degree of local order, solid-state NMR spectra of such systems exhibit an unusually high level of resolution, rendering them an ideal target for solid-state NMR investigations. Recently, our group has solved the structure of one particular supramolecular assembly, the type-iii-secretion-system needle. The needle subunit comprises around 80 residues. Many interesting supramolecular assemblies with unknown structure have subunits larger in size, which requires development of tailored solid-state NMR strategies to address their structures. In this "Perspective" article, we provide a view on different approaches to enhance sensitivity and resolution in biological solid-state NMR with a focus on the possible application to supramolecular assemblies with large subunit sizes.

Atomic-resolution structure of cytoskeletal bactofilin by solid-state NMR
Shi, C., Fricke, P., Lin(*), L., Chevelkov, V., Wegstroth(*), M., Giller(*), K., Becker(*), S., Thanbichler(*), M.; Lange, A.
Sci Adv, 1:e1501087
(2015)

Tags: Molecular Biophysics (Lange, A.)

Abstract: Bactofilins are a recently discovered class of cytoskeletal proteins of which no atomic-resolution structure has been reported thus far. The bacterial cytoskeleton plays an essential role in a wide range of processes, including morphogenesis, cell division, and motility. Among the cytoskeletal proteins, the bactofilins are bacteria-specific and do not have a eukaryotic counterpart. The bactofilin BacA of the species Caulobacter crescentus is not amenable to study by x-ray crystallography or solution nuclear magnetic resonance (NMR) because of its inherent noncrystallinity and insolubility. We present the atomic structure of BacA calculated from solid-state NMR-derived distance restraints. We show that the core domain of BacA forms a right-handed beta helix with six windings and a triangular hydrophobic core. The BacA structure was determined to 1.0 A precision (heavy-atom root mean square deviation) on the basis of unambiguous restraints derived from four-dimensional (4D) HN-HN and 2D C-C NMR spectra.

In vivo visualization of osteoarthritic hypertrophic lesions
Hu(*), H. Y., Lim(*), N. H., Juretschke(*), H. P., Ding-Pfennigdorff(*), D., Florian(*), P., Kohlmann(*), M., Kandira(*), A., von Kries(*), J. P., Saas(*), J., Rudolphi(*), K. A., Wendt(*), K. U., Nagase(*), H., Plettenburg(*), O., Nazare, M.; Schultz(*), C.
Chem Sci, 6:6256-6261
(2015)

Tags: Medicinal Chemistry (Nazare)

Abstract: Osteoarthritis (OA) is one of the most common diseases in the aging population. While disease progress in humans is monitored indirectly by X-ray or MRI, small animal OA lesions detection always requires surgical intervention and histology. Here we introduce bimodal MR/NIR probes based on cartilage-targeting 1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid amide (DOTAM) that are directly administered to the joint cavity. We demonstrate applications in healthy and diseased rat joints by MRI in vivo. The same joints are inspected post-mortem by fluorescence microscopy, showing not only the precise location of the reagents but also revealing details such as focal cartilage damage and chondrophyte or osteophyte formation. This allows for determining the distinct pathological state of the disease and the regeneration capability of the animal model and will help to correctly assess the effect of potential disease modifying OA drugs (DMOADs) in the future.

Disruption of adaptor protein 2mu (AP-2mu) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing
Jung(*), S., Maritzen, T., Wichmann(*), C., Jing(*), Z., Neef(*), A., Revelo(*), N. H., Al-Moyed(*), H., Meese(*), S., Wojcik(*), S. M., Panou(*), I., Bulut(*), H., Schu(*), P., Ficner(*), R., Reisinger(*), E., Rizzoli(*), S. O., Neef(*), J., Strenzke(*), N., Haucke, V.; Moser(*), T.
EMBO J, 34:2686-2702
(2015)

Tags: Membrane Traffic and Cell Motility (Maritzen), Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2mu (AP-2mu) is required for release site replenishment and hearing. We show that hair cell-specific disruption of AP-2mu slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane-proximal vesicles and intact endocytic membrane retrieval. Sound-driven postsynaptic spiking was reduced in a use-dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosome-like vacuoles, fewer clathrin-coated endocytic intermediates, and vesicle depletion of the membrane-distal synaptic ribbon in AP-2mu-deficient IHCs, indicating a further role of AP-2mu in clathrin-dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP-2 sorts its IHC-cargo otoferlin. We propose that binding of AP-2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP-2 in synaptic vesicle reformation.

Solid-state NMR Study of the YadA Membrane-Anchor Domain in the Bacterial Outer Membrane
Shahid, S. A., Nagaraj, M., Chauhan(*), N., Franks, T. W., Bardiaux(*), B., Habeck(*), M., Orwick-Rydmark(*), M., Linke(*), D.; van Rossum, B. J.
Angew Chem Int Ed Engl, 54:12602-12606
(2015)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: MAS-NMR was used to study the structure and dynamics at ambient temperatures of the membrane-anchor domain of YadA (YadA-M) in a pellet of the outer membrane of E. coli in which it was expressed. YadA is an adhesin from the pathogen Yersinia enterocolitica that is involved in interactions with the host cell, and it is a model protein for studying the autotransport process. Existing assignments were sucessfully transferred to a large part of the YadA-M protein in the E. coli lipid environment by using (13) C-(13) C DARR and PDSD spectra at different mixing times. The chemical shifts in most regions of YadA-M are unchanged relative to those in microcrystalline YadA-M preparations from which a structure has previously been solved, including the ASSA region that is proposed to be involved in transition-state hairpin formation for transport of the soluble domain. Comparisons of the dynamics between the microcrystalline and membrane-embedded samples indicate greater flexibility of the ASSA region in the outer-membrane preparation at physiological temperatures. This study will pave the way towards MAS-NMR structure determination of membrane proteins, and a better understanding of functionally important dynamic residues in native membrane environments.

In Vivo Evidence for Lysosome Depletion and Impaired Autophagic Clearance in Hereditary Spastic Paraplegia Type SPG11
Varga(*), R. E., Khundadze(*), M., Damme(*), M., Nietzsche(*), S., Hoffmann(*), B., Stauber, T., Koch(*), N., Hennings(*), J. C., Franzka(*), P., Huebner(*), A. K., Kessels(*), M. M., Biskup(*), C., Jentsch, T. J., Qualmann(*), B., Braulke(*), T., Kurth(*), I., Beetz(*), C.; Hübner(*), C. A.
Plos Genet, 11:e1005454
(2015)

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs). Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice.

Specific binding of Clostridium perfringens enterotoxin fragment to Claudin-b and modulation of zebrafish epidermal barrier
Zhang(*), J., Ni(*), C., Yang(*), Z., Piontek, A., Chen(*), H., Wang(*), S., Fan(*), Y., Qin(*), Z.; Piontek(*), J.
Exp Dermatol, 24:605-610
(2015)

Tags: Structural Bioinformatics and Protein Design (Krause, G.)

Abstract: Claudins (Cldn) are the major components of tight junctions (TJs) sealing the paracellular cleft in tissue barriers of various organs. Zebrafish Cldnb, the homolog of mammalian Cldn4, is expressed at epithelial cell-cell contacts and is important for regulating epidermal permeability. The bacterial toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to a subset of mammalian Cldns. In this study, we used the Cldn-binding C-terminal domain of CPE (194-319 amino acids, cCPE 194-319 ) to investigate its functional role in modulating zebrafish larval epidermal barriers. In vitro analyses show that cCPE 194-319 removed Cldn4 from epithelial cells and disrupted the monolayer tightness, which could be rescued by the removal of cCPE 194-319. Incubation of zebrafish larvae with cCPE 194-319 removed Cldnb specifically from the epidermal cell membrane. Dye diffusion analysis with 4-kDa fluorescent dextran indicated that the permeability of the epidermal barrier increased due to cCPE 194-319 incubation. Electron microscopic investigation revealed reversible loss of TJ integrity by Cldnb removal. Collectively, these results suggest that cCPE 194-319 could be used as a Cldnb modulator to transiently open the epidermal barrier in zebrafish. In addition, zebrafish might be used as an in vivo system to investigate the capability of cCPE to enhance drug delivery across tissue barriers.

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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
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info(at)fmp-berlin.de

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