FMP Publications

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

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Defining a conformational consensus motif in cotransin-sensitive signal sequences: a proteomic and site-directed mutagenesis study
Klein, W., Westendorf, C., Schmidt, A., Conill-Cortes, M., Rutz, C., Blohs, M., Beyermann, M., Protze, J., Krause, G., Krause, E.; Schülein, R.
Plos One, 10:e0120886

Tags: Protein Trafficking (Schülein), Mass Spectrometry (Krause, E.), Structural Bioinformatics and Protein Design (Krause, G.), Peptide Chemistry (Beyermann)

Abstract: The cyclodepsipeptide cotransin was described to inhibit the biosynthesis of a small subset of proteins by a signal sequence-discriminatory mechanism at the Sec61 protein-conducting channel. However, it was not clear how selective cotransin is, i.e. how many proteins are sensitive. Moreover, a consensus motif in signal sequences mediating cotransin sensitivity has yet not been described. To address these questions, we performed a proteomic study using cotransin-treated human hepatocellular carcinoma cells and the stable isotope labelling by amino acids in cell culture technique in combination with quantitative mass spectrometry. We used a saturating concentration of cotransin (30 micromolar) to identify also less-sensitive proteins and to discriminate the latter from completely resistant proteins. We found that the biosynthesis of almost all secreted proteins was cotransin-sensitive under these conditions. In contrast, biosynthesis of the majority of the integral membrane proteins was cotransin-resistant. Cotransin sensitivity of signal sequences was neither related to their length nor to their hydrophobicity. Instead, in the case of signal anchor sequences, we identified for the first time a conformational consensus motif mediating cotransin sensitivity.

Assembly and function of claudins: Structure-function relationships based on homology models and crystal structures
Krause, G., Protze, J.; Piontek(*), J.
Semin Cell Dev Biol, 42:3-12

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

Abstract: The tetra-span transmembrane proteins of the claudin family are critical components of formation and function of tight junctions (TJ). Homo- and heterophilic side-by-side (cis) and intercellular head-to-head (trans) interactions of 27 claudin-subtypes regulate tissue-specifically the paracellular permeability and/or tightness between epithelial or endothelial cells. This review highlights the functional impact that has been identified for particular claudin residues by relating them to structural features and architectural characteristics in the light of structural advances, which have been contributed by homology models, cryo-electron microscopy and crystal structures. The differing contributions to the TJ functionalities by claudins are dissected for the transmembrane region, the first and the second extracellular loop of claudins separately. Their particular impact to oligomerisation and TJ strand- and pore-formation is surveyed. Detailed knowledge about structure-function relationships about claudins helps to reveal the molecular mechanisms of TJ assembly and regulation of paracellular permeability, which is yet not fully understood.

Directed structural modification of Clostridium perfringens enterotoxin to enhance binding to claudin-5
Protze, J., Eichner, M., Piontek, A., Dinter, S., Rossa, J., Blecharz(*), K. G., Vajkoczy(*), P., Piontek(*), J.; Krause, G.
Cellular and molecular life sciences : CMLS, 72:1417-1432

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

Abstract: Clostridium perfringens enterotoxin (CPE) binds to distinct claudins (Clds), which regulate paracellular barrier functions in endo- and epithelia. The C-terminal domain (cCPE) has the potential for selective claudin modulation, since it only binds to a subset of claudins, e.g., Cld3 and Cld4 (cCPE receptors). Cld5 (non-CPE receptor) is a main constituent in tight junctions (TJ) of the blood-brain barrier. We aimed to reveal claudin recognition mechanisms of cCPE and to create a basis for a Cld5-binder. By utilizing structure-based interaction models, mutagenesis and assays of cCPE-binding to the TJ-free cell line HEK293, transfected with human Cld1 and murine Cld5, we showed how cCPE-binding to Cld1 and Cld5 is prevented by two residues in extracellular loop 2 of Cld1 (Asn(150) and Thr(153)) and Cld5 (Asp(149) and Thr(151)). Binding to Cld5 is especially attenuated by the lack of a bulky hydrophobic residue like leucine at position 151. By downsizing the binding pocket and compensating for the lack of this leucine residue, we created a novel cCPE-variant; cCPEY306W/S313H binds Cld5 with nanomolar affinity (K d 33 +/- 10 nM). Finally, the effective binding to endogenously Cld5-expressing blood-brain barrier model cells (murine microvascular endothelial cEND cell line) suggests cCPEY306W/S313H as basis for Cld5-specific modulation to improve paracellular drug delivery, or to target claudin overexpressing tumors.

Sensitivity and resolution of proton detected spectra of a deuterated protein at 40 and 60 kHz magic-angle-spinning
Nieuwkoop, A. J., Franks, W. T., Rehbein, K., Diehl, A., Akbey, Ü., Engelke(*), F., Emsley(*), L., Pintacuda(*), G.; Oschkinat, H.
J Biomol NMR, 61:161-171

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: The use of small rotors capable of very fast magic-angle spinning (MAS) in conjunction with proton dilution by perdeuteration and partial reprotonation at exchangeable sites has enabled the acquisition of resolved, proton detected, solid-state NMR spectra on samples of biological macromolecules. The ability to detect the high-gamma protons, instead of carbons or nitrogens, increases sensitivity. In order to achieve sufficient resolution of the amide proton signals, rotors must be spun at the maximum rate possible given their size and the proton back-exchange percentage tuned. Here we investigate the optimal proton back-exchange ratio for triply labeled SH3 at 40 kHz MAS. We find that spectra acquired on 60 % back-exchanged samples in 1.9 mm rotors have similar resolution at 40 kHz MAS as spectra of 100 % back-exchanged samples in 1.3 mm rotors spinning at 60 kHz MAS, and for (H)NH 2D and (H)CNH 3D spectra, show 10-20 % higher sensitivity. For 100 % back-exchanged samples, the sensitivity in 1.9 mm rotors is superior by a factor of 1.9 in (H)NH and 1.8 in (H)CNH spectra but at lower resolution. For (H)C(C)NH experiments with a carbon-carbon mixing period, this sensitivity gain is lost due to shorter relaxation times and less efficient transfer steps. We present a detailed study on the sensitivity of these types of experiments for both types of rotors, which should enable experimentalists to make an informed decision about which type of rotor is best for specific applications.

Phosphoinositides in endocytosis
Posor, Y., Eichhorn-Grünig, M.; Haucke, V.
Biochim Biophys Acta, 1851:794-804

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: The internalization and subsequent endosomal trafficking of proteins and membrane along the endocytic pathway is a fundamental cellular process. Over the last two decades, this pathway has emerged to be subject to extensive regulation by phosphoinositides (PIs), phosphorylated derivatives of the minor membrane phospholipid phosphatidylinositol. Clathrin-mediated endocytosis (CME) is the endocytic mechanism characterized in most detail. It now represents a prime example of a process spatiotemporally organized by the interplay of PI metabolizing enzymes. The most abundant PI, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)], serves as a denominator of plasma membrane identity and together with cargo proteins is instrumental for the initiation of clathrin-coated pit (CCP) formation. During later stages of the process, the generation of phosphatidylinositol-3,4-bisphosphate [PI(3,4)P(2)] and the dephosphorylation of PI(4,5)P(2)regulate CCP maturation and vesicle uncoating. Here we provide an overview of the mechanisms by which PIs are made and consumed to regulate CME and other endocytic pathways and how conversion of PIs en route to endosomes may be accomplished. Mutations in PI converting enzymes are linked to multiple diseases ranging from mental retardation and neurodegeneration, to inherited muscle and kidney disorders suggesting that the tight control of PI levels along the endocytic pathway plays a critical role in cell physiology. This article is part of a Special Issue entitled Phosphoinositides.

Muscular Dystrophy Mutations Impair the Nuclear Envelope Emerin Self-assembly Properties
Herrada(*), I., Samson(*), C., Velours(*), C., Renault(*), L., Ostlund(*), C., Chervy(*), P., Puchkov, D., Worman(*), H. J., Buendia(*), B.; Zinn-Justin(*), S.
ACS Chem Biol, 10:2733-2742

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: More than 100 genetic mutations causing X-linked Emery-Dreifuss muscular dystrophy have been identified in the gene encoding the integral inner nuclear membrane protein emerin. Most mutations are nonsense or frameshift mutations that lead to the absence of emerin in cells. Only very few cases are due to missense or short in-frame deletions. Molecular mechanisms explaining the corresponding emerin variants' loss of function are particularly difficult to identify because of the mostly intrinsically disordered state of the emerin nucleoplasmic region. We now demonstrate that this EmN region can be produced as a disordered monomer, as revealed by nuclear magnetic resonance, but rapidly self-assembles in vitro. Increases in concentration and temperature favor the formation of long curvilinear filaments with diameters of approximately 10 nm, as observed by electron microscopy. Assembly of these filaments can be followed by fluorescence through Thioflavin-T binding and by Fourier-transform Infrared spectrometry through formation of beta-structures. Analysis of the assembly properties of five EmN variants reveals that del95-99 and Q133H impact filament assembly capacities. In cells, these variants are located at the nuclear envelope, but the corresponding quantities of emerin-emerin and emerin-lamin proximities are decreased compared to wild-type protein. Furthermore, variant P183H favors EmN aggregation in vitro, and variant P183T provokes emerin accumulation in cytoplasmic foci in cells. Substitution of residue Pro183 might systematically favor oligomerization, leading to emerin aggregation and mislocalization in cells. Our results suggest that emerin self-assembly is necessary for its proper function and that a loss of either the protein itself or its ability to self-assemble causes muscular dystrophy.

Overlapping functions of stonin 2 and SV2 in sorting of the calcium sensor synaptotagmin 1 to synaptic vesicles
Kaempf, N., Kochlamazashvili, G., Puchkov, D., Maritzen, T., Bajjalieh(*), S. M., Kononenko, N. L.; Haucke, V.
Proc Natl Acad Sci U S A, 112:7297-7302

Tags: Molecular Pharmacology and Cell Biology (Haucke), Membrane Traffic and Cell Motility (Maritzen), Behavioral Neurodynamics (Korotkova/Ponomarenko)

Abstract: Neurotransmission involves the calcium-regulated exocytic fusion of synaptic vesicles (SVs) and the subsequent retrieval of SV membranes followed by reformation of properly sized and shaped SVs. An unresolved question is whether each SV protein is sorted by its own dedicated adaptor or whether sorting is facilitated by association between different SV proteins. We demonstrate that endocytic sorting of the calcium sensor synaptotagmin 1 (Syt1) is mediated by the overlapping activities of the Syt1-associated SV glycoprotein SV2A/B and the endocytic Syt1-adaptor stonin 2 (Stn2). Deletion or knockdown of either SV2A/B or Stn2 results in partial Syt1 loss and missorting of Syt1 to the neuronal surface, whereas deletion of both SV2A/B and Stn2 dramatically exacerbates this phenotype. Selective missorting and degradation of Syt1 in the absence of SV2A/B and Stn2 impairs the efficacy of neurotransmission at hippocampal synapses. These results indicate that endocytic sorting of Syt1 to SVs is mediated by the overlapping activities of SV2A/B and Stn2 and favor a model according to which SV protein sorting is guarded by both cargo-specific mechanisms as well as association between SV proteins.

Vesicular Synaptobrevin/VAMP2 Levels Guarded by AP180 Control Efficient Neurotransmission
Koo, S. J., Kochlamazashvili, G., Rost(*), B., Puchkov, D., Gimber, N., Lehmann, M., Tadeus, G., Schmoranzer, J., Rosenmund(*), C., Haucke, V.; Maritzen, T.
Neuron, 88:330-344

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

Abstract: Neurotransmission depends on synaptic vesicle (SV) exocytosis driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation of vesicular synaptobrevin/VAMP2 (Syb2). Exocytic fusion is followed by endocytic SV membrane retrieval and the high-fidelity reformation of SVs. Syb2 is the most abundant SV protein with 70 copies per SV, yet, one to three Syb2 molecules appear to be sufficient for basal exocytosis. Here we demonstrate that loss of the Syb2-specific endocytic adaptor AP180 causes a moderate activity-dependent reduction of vesicular Syb2 levels, defects in SV reformation, and a corresponding impairment of neurotransmission that lead to excitatory/inhibitory imbalance, epileptic seizures, and premature death. Further reduction of Syb2 levels in AP180(-/-)/Syb2(+/-) mice results in perinatal lethality, whereas Syb2(+/-) mice partially phenocopy loss of AP180, indicating that reduced vesicular Syb2 levels underlie the observed defects in neurotransmission. Thus, a large vesicular Syb2 pool maintained by AP180 is crucial to sustain efficient neurotransmission and SV reformation.

Multicolor Caged dSTORM Resolves the Ultrastructure of Synaptic Vesicles in the Brain
Lehmann, M., Gottschalk, B., Puchkov, D., Schmieder, P., Schwagerus, S., Hackenberger, C. P., Haucke, V.; Schmoranzer, J.
Angew Chem Int Ed Engl, 54:13230-13235

Tags: Molecular Pharmacology and Cell Biology (Haucke), Chemical Biology II (Hackenberger)

Abstract: The precision of single-molecule localization-based super-resolution microscopy, including dSTORM, critically depends on the number of detected photons per localization. Recently, reductive caging of fluorescent dyes followed by UV-induced recovery in oxidative buffer systems was used to increase the photon yield and thereby the localization precision in single-color dSTORM. By screening 39 dyes for their fluorescence caging and recovery kinetics, we identify novel dyes that are suitable for multicolor caged dSTORM. Using a dye pair suited for registration error-free multicolor dSTORM based on spectral demixing (SD), a multicolor localization precision below 15 nm was achieved. Caged SD-dSTORM can resolve the ultrastructure of single 40 nm synaptic vesicles in brain sections similar to images obtained by immuno-electron microscopy, yet with much improved label density in two independent channels.

Vesicle uncoating regulated by SH3-SH3 domain-mediated complex formation between endophilin and intersectin at synapses
Pechstein, A., Gerth(*), F., Milosevic(*), I., Jäpel, M., Eichhorn-Grünig, M., Vorontsova(*), O., Bacetic, J., Maritzen, T., Shupliakov(*), O., Freund(*), C.; Haucke, V.
Embo Rep, 16:232-239

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

Abstract: Neurotransmission involves the exo-endocytic cycling of synaptic vesicle (SV) membranes. Endocytic membrane retrieval and clathrin-mediated SV reformation require curvature-sensing and membrane-bending BAR domain proteins such as endophilin A. While their ability to sense and stabilize curved membranes facilitates membrane recruitment of BAR domain proteins, the precise mechanisms by which they are targeted to specific sites of SV recycling has remained unclear. Here, we demonstrate that the multi-domain scaffold intersectin 1 directly associates with endophilin A to facilitate vesicle uncoating at synapses. Knockout mice deficient in intersectin 1 accumulate clathrin-coated vesicles at synapses, a phenotype akin to loss of endophilin function. Intersectin 1/endophilin A1 complex formation is mediated by direct binding of the SH3B domain of intersectin to a non-canonical site on the SH3 domain of endophilin A1. Consistent with this, intersectin-binding defective mutant endophilin A1 fails to rescue clathrin accumulation at neuronal synapses derived from endophilin A1-3 triple knockout (TKO) mice. Our data support a model in which intersectin aids endophilin A recruitment to sites of clathrin-mediated SV recycling, thereby facilitating vesicle uncoating.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
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