FMP Publications

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

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References
AKAP18:PKA-RIIalpha structure reveals crucial anchor points for recognition of regulatory subunits of PKA
Götz, F., Roske(*), Y., Schulz(*), M. S., Autenrieth(*), K., Bertinetti(*), D., Faelber(*), K., Zühlke(*), K., Kreuchwig, A., Kennedy(*), E. J., Krause, G., Daumke(*), O., Herberg(*), F. W., Heinemann(*), U.; Klussmann(*), E.
Biochem J, 473:1881-1894
(2016)

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

Abstract: A-kinase anchoring proteins (AKAPs) interact with the dimerization/docking (D/D) domains of regulatory subunits of the ubiquitous protein kinase A (PKA). AKAPs tether PKA to defined cellular compartments establishing distinct pools to increase the specificity of PKA signalling. Here, we elucidated the structure of an extended PKA-binding domain of AKAP18beta bound to the D/D domain of the regulatory RIIalpha subunits of PKA. We identified three hydrophilic anchor points in AKAP18beta outside the core PKA-binding domain, which mediate contacts with the D/D domain. Such anchor points are conserved within AKAPs that bind regulatory RII subunits of PKA. We derived a different set of anchor points in AKAPs binding regulatory RI subunits of PKA. In vitro and cell-based experiments confirm the relevance of these sites for the interaction of RII subunits with AKAP18 and of RI subunits with the RI-specific smAKAP. Thus we report a novel mechanism governing interactions of AKAPs with PKA. The sequence specificity of each AKAP around the anchor points and the requirement of these points for the tight binding of PKA allow the development of selective inhibitors to unequivocally ascribe cellular functions to the AKAP18-PKA and other AKAP-PKA interactions.

Antifungal membranolytic activity of the tyrocidines against filamentous plant fungi
Rautenbach(*), M., Troskie(*), A. M., Vosloo(*), J. A.; Dathe, M. E.
Biochimie, 130:122-131
(2016)

Tags: Peptide-Lipid-Interaction/ Peptide Transport (Dathe)

Abstract: The tyrocidines and analogues are cyclic decapeptides produced by Brevibacillus parabrevis with a conserved sequence of cyclo(D-Phe1-Pro2-X3-x4-Asn5-Gln6-X7-Val8-X9-Leu10) with Trp3,4/Phe3,4 in the aromatic dipeptide unit, Lys9/Orn9 as their cationic residue and Tyr (tyrocidines), Trp (tryptocidines) or Phe (phenicidines) in position 7. Previous studies indicated they have a broad antifungal spectrum with the peptides containing a Tyr residue in position 7 being more active than those with a Phe or Trp residue in this position. Detailed analysis of antifungal inhibition parameters revealed that Phe3-D-Phe4 in the aromatic dipeptide unit lead to more consistent activity against the three filamentous fungi in this study. These peptides exhibited high membrane activity and fast leakage kinetics against model membranes emulating fungal membranes, with selectivity towards ergosterol containing membranes. More fluid membranes and doping of liposomes with the sphingolipid, glucosylceramide, led to a decreased permeabilising activity. Peptide-induced uptake of membrane impermeable dyes was observed in hyphae of both Fusarium solani and Botrytis cinerea, with uptake more pronounced at the hyphal growth tips that are known to contain ergosterol-sphigolipid rich lipid rafts. Tyrocidine interaction with these rafts may lead to the previously observed fungal hyperbranching. However, the leakage of model membranes and Bot. cinerea did not correlate directly with the antifungal inhibition parameters, indicating another target or mode of action. Proteinase K treatment of target fungi had a minimal influence or even improved the tyrocidine activity, ruling out a mannoprotein target in the fungal cell wall. beta-glucanase treatment of Bot. cinerea did not significantly affect the tyrocidine activity, but there was a significant loss in activity towards the beta-glucanase treated F. solani. This study showed the tyrocidine antifungal membrane activity is selective towards ergosterol and possibly lipid rafts, but also point to additional targets such as the cell wall beta-glucans that could modulate their activity.

5-Aryl-2-(naphtha-1-yl)sulfonamido-thiazol-4(5H)-ones as clathrin inhibitors
Robertson(*), M. J., Horatscheck, A., Sauer, S., von Kleist(*), L., Baker, J. R., Stahlschmidt, W., Nazare, M., Whiting(*), A., Chau(*), N., Robinson(*), P. J., Haucke, V.; McCluskey(*), A.
Org Biomol Chem, 14:11266-11278
(2016)

Tags: Molecular Pharmacology and Cell Biology (Haucke), Medicinal Chemistry (Nazare)

Abstract: The development of a (Z)-5-((6,8-dichloro-4-oxo-4H-chromen-3-yl)methylene)-2-thioxothiazolidin-4-one (2), rhodanine-based lead that led to the Pitstop(R) 2 family of clathrin inhibitors is described herein. Head group substitution and bioisosteric replacement of the rhodanine core with a 2-aminothiazol-4(5H)-one scaffold eliminated off target dynamin activity. A series of N-substituents gave first phenylglycine (20, IC50 approximately 20 muM) then phenyl (25, IC50 approximately 7.1 muM) and 1-napthyl sulfonamide (26, Pitstop(R) 2 compound, IC50 approximately 1.9 muM) analogues with good activity, validating this approach. A final library exploring the head group resulted in three analogues displaying either slight improvements or comparable activity (33, 38, and 29 with IC50 approximately 1.4, 1.6 and 1.8 muM respectively) and nine others with IC50 < 10 muM. These results were rationalized using in silico docking studies. Docking studies predicted enhanced Pitstop(R) 2 family binding, not a loss of binding, within the Pistop(R) groove of the reported clathrin mutant invalidating recent assumptions of poor selectivity for this family of clathrin inhibitors.

RIM-binding protein 2 regulates release probability by fine-tuning calcium channel localization at murine hippocampal synapses
Grauel(*), M. K., Maglione, M., Reddy-Alla(*), S., Willmes(*), C. G., Brockmann(*), M. M., Trimbuch(*), T., Rosenmund(*), T., Pangalos(*), M., Vardar(*), G., Stumpf(*), A., Walter, A. M., Rost(*), B. R., Eickholt(*), B. J., Haucke, V., Schmitz(*), D., Sigrist(*), S. J.; Rosenmund(*), C.
Proc Natl Acad Sci U S A, 113:11615-11620
(2016)

Tags: Molecular Pharmacology and Cell Biology (Haucke), Molecular and Theoretical Neuroscience (Walter)

Abstract: The tight spatial coupling of synaptic vesicles and voltage-gated Ca2+ channels (CaVs) ensures efficient action potential-triggered neurotransmitter release from presynaptic active zones (AZs). Rab-interacting molecule-binding proteins (RIM-BPs) interact with Ca2+ channels and via RIM with other components of the release machinery. Although human RIM-BPs have been implicated in autism spectrum disorders, little is known about the role of mammalian RIM-BPs in synaptic transmission. We investigated RIM-BP2-deficient murine hippocampal neurons in cultures and slices. Short-term facilitation is significantly enhanced in both model systems. Detailed analysis in culture revealed a reduction in initial release probability, which presumably underlies the increased short-term facilitation. Superresolution microscopy revealed an impairment in CaV2.1 clustering at AZs, which likely alters Ca2+ nanodomains at release sites and thereby affects release probability. Additional deletion of RIM-BP1 does not exacerbate the phenotype, indicating that RIM-BP2 is the dominating RIM-BP isoform at these synapses.

Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca(2+) channel-vesicle coupling
Böhme(*), M. A., Beis(*), C., Reddy-Alla(*), S., Reynolds(*), E., Mampell(*), M. M., Grasskamp, A. T., Lutzkendorf(*), J., Bergeron(*), D. D., Driller(*), J. H., Babikir(*), H., Göttfert(*), F., Robinson(*), I. M., O'Kane(*), C. J., Hell(*), S. W., Wahl(*), M. C., Stelzl(*), U., Loll(*), B., Walter, A. M.; Sigrist(*), S. J.
Nat Neurosci, 19:1311-1320
(2016)

Tags: Molecular and Theoretical Neuroscience (Walter)

Abstract: Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca(2+) channel, but molecular mechanisms controlling this are unknown. Here we found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins Syd-1 and Liprin-alpha, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca(2+) channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13A(null) mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. We identified isoform-specific Unc13-AZ scaffold interactions regulating SV-Ca(2+)-channel topology whose developmental tightening optimizes synaptic transmission.

Intracellular repair of oxidation-damaged alpha-synuclein fails to target C-terminal modification sites
Binolfi, A., Limatola, A., Verzini, S., Kosten, J., Theillet, F. X., Rose, H. M., Bekei, B., Stuiver, M., van Rossum, M.; Selenko, P.
Nat Commun, 7:10251
(2016)

Tags: In-Cell NMR (Selenko)

Abstract: Cellular oxidative stress serves as a common denominator in many neurodegenerative disorders, including Parkinson's disease. Here we use in-cell NMR spectroscopy to study the fate of the oxidation-damaged Parkinson's disease protein alpha-synuclein (alpha-Syn) in non-neuronal and neuronal mammalian cells. Specifically, we deliver methionine-oxidized, isotope-enriched alpha-Syn into cultured cells and follow intracellular protein repair by endogenous enzymes at atomic resolution. We show that N-terminal alpha-Syn methionines Met1 and Met5 are processed in a stepwise manner, with Met5 being exclusively repaired before Met1. By contrast, C-terminal methionines Met116 and Met127 remain oxidized and are not targeted by cellular enzymes. In turn, persisting oxidative damage in the C-terminus of alpha-Syn diminishes phosphorylation of Tyr125 by Fyn kinase, which ablates the necessary priming event for Ser129 modification by CK1. These results establish that oxidative stress can lead to the accumulation of chemically and functionally altered alpha-Syn in cells.

Structural disorder of monomeric alpha-synuclein persists in mammalian cells
Theillet, F. X., Binolfi, A., Bekei, B., Martorana(*), A., Rose, H. M., Stuiver, M., Verzini, S., Lorenz, D., van Rossum, M., Goldfarb(*), D.; Selenko, P.
Nature, 530:45-50
(2016)

Tags: In-Cell NMR (Selenko), Cellular Imaging (Wiesner)

Abstract: Intracellular aggregation of the human amyloid protein alpha-synuclein is causally linked to Parkinson's disease. While the isolated protein is intrinsically disordered, its native structure in mammalian cells is not known. Here we use nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy to derive atomic-resolution insights into the structure and dynamics of alpha-synuclein in different mammalian cell types. We show that the disordered nature of monomeric alpha-synuclein is stably preserved in non-neuronal and neuronal cells. Under physiological cell conditions, alpha-synuclein is amino-terminally acetylated and adopts conformations that are more compact than when in buffer, with residues of the aggregation-prone non-amyloid-beta component (NAC) region shielded from exposure to the cytoplasm, which presumably counteracts spontaneous aggregation. These results establish that different types of crowded intracellular environments do not inherently promote alpha-synuclein oligomerization and, more generally, that intrinsic structural disorder is sustainable in mammalian cells.

Bimodal antagonism of PKA signalling by ARHGAP36
Eccles(*), R. L., Czajkowski(*), M. T., Barth(*), C., Müller(*), P. M., McShane(*), E., Grunwald(*), S., Beaudette(*), P., Mecklenburg(*), N., Volkmer, R., Zühlke(*), K., Dittmar(*), G., Selbach(*), M., Hammes(*), A., Daumke(*), O., Klussmann(*), E., Urbe(*), S.; Rocks(*), O.
Nat Commun, 7:12963
(2016)

Tags: Peptide Synthesis (Hackenberger/Volkmer)

Abstract: Protein kinase A is a key mediator of cAMP signalling downstream of G-protein-coupled receptors, a signalling pathway conserved in all eukaryotes. cAMP binding to the regulatory subunits (PKAR) relieves their inhibition of the catalytic subunits (PKAC). Here we report that ARHGAP36 combines two distinct inhibitory mechanisms to antagonise PKA signalling. First, it blocks PKAC activity via a pseudosubstrate motif, akin to the mechanism employed by the protein kinase inhibitor proteins. Second, it targets PKAC for rapid ubiquitin-mediated lysosomal degradation, a pathway usually reserved for transmembrane receptors. ARHGAP36 thus dampens the sensitivity of cells to cAMP. We show that PKA inhibition by ARHGAP36 promotes derepression of the Hedgehog signalling pathway, thereby providing a simple rationale for the upregulation of ARHGAP36 in medulloblastoma. Our work reveals a new layer of PKA regulation that may play an important role in development and disease.

Sulindac Sulfide Induces the Formation of Large Oligomeric Aggregates of the Alzheimer's Disease Amyloid-beta Peptide Which Exhibit Reduced Neurotoxicity
Prade(*), E., Barucker(*), C., Sarkar(*), R., Althoff-Ospelt(*), G., Lopez del Amo, J. M., Hossain(*), S., Zhong(*), Y., Multhaup(*), G.; Reif(*), B.
Biochemistry, 55:1839-1849
(2016)

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: Alzheimer's disease is characterized by deposition of the amyloid beta-peptide (Abeta) in brain tissue of affected individuals. In recent years, many potential lead structures have been suggested that can potentially be used for diagnosis and therapy. However, the mode of action of these compounds is so far not understood. Among these small molecules, the nonsteroidal anti-inflammatory drug (NSAID) sulindac sulfide received a lot of attention. In this manuscript, we characterize the interaction between the monomeric Abeta peptide and the NSAID sulindac sulfide. We find that sulindac sulfide efficiently depletes the pool of toxic oligomers by enhancing the rate of fibril formation. In vitro, sulindac sulfide forms colloidal particles which catalyze the formation of fibrils. Aggregation is immediate, presumably by perturbing the supersaturated Abeta solution. We find that sulindac sulfide induced Abeta aggregates are structurally homogeneous. The C-terminal part of the peptide adopts a beta-sheet structure, whereas the N-terminus is disordered. The salt bridge between D23 and K28 is present, similar as in wild type fibril structures. (13)C-(19)F transferred echo double resonance experiments suggest that sulindac sulfide colocalizes with the Abeta peptide in the aggregate.

Semi-synthesis of a tag-free O-GlcNAcylated tau protein by sequential chemoselective ligation
Schwagerus, S., Reimann, O., Despres(*), C., Smet-Nocca(*), C.; Hackenberger, C. P.
J Pept Sci, 22:327-333
(2016)

Tags: Chemical Biology II (Hackenberger)

Abstract: In this paper, the first semi-synthesis of the Alzheimer-relevant tau protein carrying an O-GlcNAcylation is demonstrated by using sequential chemoselective ligation. The 52-amino acid C-terminus of tau was obtained by native chemical ligation between two synthetic peptide fragments, one carrying the O-GlcNAc moiety on Ser400, which has recently been demonstrated to inhibit tau phosphorylation and to hinder tau oligomerization, and the other equipped with a photocleavable biotin handle. After desulfurization to deliver a native alanine at the ligation junction, the N-terminal cysteine was unmasked, and the peptide was further used for expressed protein ligation to generate the full-length tau protein, which was purified by a photocleavable biotin tag. We thus provide a synthetic route to obtain a homogenous tag-free O-GlcNAcylated tau protein that can further help to elucidate the significance of posttranslational modification on the tau protein and pave the way for evaluating possible drug targets in Alzheimer's disease. Copyright (c) 2016 European Peptide Society and John Wiley & Sons, Ltd.

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