FMP Publications

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

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References
Fast neurotransmitter release regulated by the endocytic scaffold intersectin
Sakaba(*), T., Kononenko, N. L., Bacetic, J., Pechstein, A., Schmoranzer, J., Yao(*), L., Barth(*), H., Shupliakov(*), O., Kobler(*), O., Aktories(*), K.; Haucke, V.
Proc Natl Acad Sci U S A, 110:8266-8271
(2013)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Sustained fast neurotransmission requires the rapid replenishment of release-ready synaptic vesicles (SVs) at presynaptic active zones. Although the machineries for exocytic fusion and for subsequent endocytic membrane retrieval have been well characterized, little is known about the mechanisms underlying the rapid recruitment of SVs to release sites. Here we show that the Down syndrome-associated endocytic scaffold protein intersectin 1 is a crucial factor for the recruitment of release-ready SVs. Genetic deletion of intersectin 1 expression or acute interference with intersectin function inhibited the replenishment of release-ready vesicles, resulting in short-term depression, without significantly affecting the rate of endocytic membrane retrieval. Acute perturbation experiments suggest that intersectin-mediated vesicle replenishment involves the association of intersectin with the fissioning enzyme dynamin and with the actin regulatory GTPase CDC42. Our data indicate a role for the endocytic scaffold intersectin in fast neurotransmitter release, which may be of prime importance for information processing in the brain.

Inhibition of clathrin by pitstop 2 activates the spindle assembly checkpoint and induces cell death in dividing HeLa cancer cells
Smith(*), C. M., Haucke, V., McCluskey(*), A., Robinson(*), P. J.; Chircop(*), M.
Mol Cancer, 12
(2013)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Background: During metaphase clathrin stabilises the mitotic spindle kinetochore(K)-fibres. Many anti-mitotic compounds target microtubule dynamics. Pitstop 2 (TM) is the first small molecule inhibitor of clathrin terminal domain and inhibits clathrin-mediated endocytosis. We investigated its effects on a second function for clathrin in mitosis. Results: Pitstop 2 did not impair clathrin recruitment to the spindle but disrupted its function once stationed there. Pitstop 2 trapped HeLa cells in metaphase through loss of mitotic spindle integrity and activation of the spindle assembly checkpoint, phenocopying clathrin depletion and aurora A kinase inhibition. Conclusions: Pitstop 2 is therefore a new tool for investigating clathrin spindle dynamics. Pitstop 2 reduced viability in dividing HeLa cells, without affecting dividing non-cancerous NIH3T3 cells, suggesting that clathrin is a possible novel anti-mitotic drug target.

PI4K2beta/AP-1-based TGN-endosomal sorting regulates Wnt signaling
Wieffer, M., Cibrian Uhalte(*), E., Posor, Y., Otten(*), C., Branz, K., Schütz, I., Mössinger, J., Schu(*), P., Abdelilah-Seyfried(*), S., Krauss, M.; Haucke, V.
Curr Biol, 23:2185-2190
(2013)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Endosomal membrane traffic serves crucial roles in cell physiology, signaling, and development. Sorting between endosomes and the trans-Golgi network (TGN) is regulated among other factors by the adaptor AP-1, an essential component of multicellular organisms. Membrane recruitment of AP-1 requires phosphatidylinositol 4-phosphate [PI(4)P], though the precise mechanisms and PI4 kinase isozyme (or isozymes) involved in generation of this PI(4)P pool remain unclear. The Wnt pathway is a major developmental signaling cascade and depends on endosomal sorting in Wnt-sending cells. Whether TGN/endosomal sorting modulates signaling downstream of Frizzled (Fz) receptors in Wnt-receiving cells is unknown. Here, we identify PI4-kinase type 2beta (PI4K2beta) as a regulator of TGN/endosomal sorting and Wnt signaling. PI4K2beta and AP-1 interact directly and are required for efficient sorting between endosomes and the TGN. Zebrafish embryos depleted of PI4K2beta or AP-1 lack pectoral fins due to defective Wnt signaling. Rescue experiments demonstrate requirements for PI4K2beta-AP-1 complex formation and PI4K2beta-mediated PI(4)P synthesis. Furthermore, PI4K2beta binds to the Fz-associated component Dishevelled (Dvl) and regulates endosomal recycling of Fz receptors and Wnt target gene expression. These data reveal an evolutionarily conserved role for PI4K2beta and AP-1 in coupling phosphoinositide metabolism to AP-1-mediated sorting and Wnt signaling.

The Bacterial Translocon SecYEG Opens upon Ribosome Binding
Knyazev(*), D. G., Lents(*), A., Krause, E., Ollinger(*), N., Siligan(*), C., Papinski(*), D., Winter(*), L., Horner(*), A.; Pohl(*), P.
Journal of Biological Chemistry, 288:17941-17946
(2013)

Tags: Mass Spectrometry (Krause, E.)

Abstract: In co-translational translocation, the ribosome funnel and the channel of the protein translocation complex SecYEG are aligned. For the nascent chain to enter the channel immediately after synthesis, a yet unidentified signal triggers displacement of the SecYEG sealing plug from the pore. Here, we show that ribosome binding to the resting SecYEG channel triggers this conformational transition. The purified and reconstituted SecYEG channel opens to form a large ion-conducting channel, which has the conductivity of the plug deletion mutant. The number of ion-conducting channels inserted into the planar bilayer per fusion event roughly equals the number of SecYEG channels counted by fluorescence correlation spectroscopy in a single proteoliposome. Thus, the open probability of the channel must be close to unity. To prevent the otherwise lethal proton leak, a closed post-translational conformation of the SecYEG complex bound to a ribosome must exist.

Vestibular role of KCNQ4 and KCNQ5 K+ channels revealed by mouse models
Spitzmaul, G., Tolosa(*), L., Winkelman(*), B. H., Heidenreich, M., Frens(*), M. A., Chabbert(*), C., De Zeeuw(*), C. I.; Jentsch, T. J.
J Biol Chem, 288:9334-9344
(2013)

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: The function of sensory hair cells of the cochlea and vestibular organs depends on an influx of K(+) through apical mechanosensitive ion channels and its subsequent removal over their basolateral membrane. The KCNQ4 (Kv7.4) K(+) channel, which is mutated in DFNA2 human hearing loss, is expressed in the basal membrane of cochlear outer hair cells where it may mediate K(+) efflux. Like the related K(+) channel KCNQ5 (Kv7.5), KCNQ4 is also found at calyx terminals ensheathing type I vestibular hair cells where it may be localized pre- or postsynaptically. Making use of Kcnq4(-/-) mice lacking KCNQ4, as well as Kcnq4(dn/dn) and Kcnq5(dn/dn) mice expressing dominant negative channel mutants, we now show unambiguously that in adult mice both channels reside in postsynaptic calyx-forming neurons, but cannot be detected in the innervated hair cells. Accordingly, whole cell currents of vestibular hair cells did not differ between genotypes. Neither Kcnq4(-/-), Kcnq5(dn/dn) nor Kcnq4(-/-)/Kcnq5(dn/dn) double mutant mice displayed circling behavior found with severe vestibular impairment. However, a milder form of vestibular dysfunction was apparent from altered vestibulo-ocular reflexes in Kcnq4(-/-)/Kcnq5(dn/dn) and Kcnq4(-/-) mice. The larger impact of KCNQ4 may result from its preferential expression in central zones of maculae and cristae, which are innervated by phasic neurons that are more sensitive than the tonic neurons present predominantly in the surrounding peripheral zones where KCNQ5 is found. The impact of postsynaptic KCNQ4 on vestibular function may be related to K(+) removal and modulation of synaptic transmission.

In tight junctions, claudins regulate the interactions between occludin, tricellulin and marvelD3, which, inversely, modulate claudin oligomerization
Cording, J., Berg, J., Käding, N., Bellmann, C., Tscheik, C., Westphal(*), J. K., Milatz(*), S., Günzel(*), D., Wolburg(*), H., Piontek, J., Huber(*), O.; Blasig, I. E.
J Cell Sci, 126:554-564
(2013)

Tags: Molecular and Cell Physiology (Blasig, IE)

Abstract: Tight junctions seal the paracellular cleft of epithelia and endothelia, form vital barriers between tissue compartments and consist of tight-junction-associated marvel proteins (TAMPs) and claudins. The function of TAMPs and the interaction with claudins are not understood. We therefore investigated the binding between the TAMPs occludin, tricellulin, and marvelD3 and their interaction with claudins in living tight-junction-free human embryonic kidney-293 cells. In contrast to claudins and occludin, tricellulin and marvelD3 showed no enrichment at cell-cell contacts indicating lack of homophilic trans-interaction between two opposing cell membranes. However, occludin, marvelD3 and tricellulin exhibited homophilic cis-interactions, along one plasma membrane, as measured by fluorescence resonance energy transfer. MarvelD3 also cis-interacted with occludin and tricellulin heterophilically. Classic claudins, such as claudin-1 to -5 may show cis-oligomerization with TAMPs, whereas the non-classic claudin-11 did not. Claudin-1 and -5 improved enrichment of occludin and tricellulin at cell-cell contacts. The low mobile claudin-1 reduced the membrane mobility of the highly mobile occludin and tricellulin, as studied by fluorescence recovery after photobleaching. Co-transfection of claudin-1 with TAMPs led to changes of the tight junction strand network of this claudin to a more physiological morphology, depicted by freeze-fracture electron microscopy. The results demonstrate multilateral interactions between the tight junction proteins, in which claudins determine the function of TAMPs and vice versa, and provide deeper insights into the tight junction assembly.

Completion of proteomic data sets by Kd measurement using cell-free synthesis of site-specifically labeled proteins
Majkut, P., Claussnitzer(*), I., Merk(*), H., Freund(*), C., Hackenberger, C. P.; Gerrits(*), M.
Plos One, 8:e82352
(2013)

Tags: Chemical Biology II (Hackenberger)

Abstract: The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (Kd values). In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST) we were able to validate interactions and to establish a binding hierarchy based on Kd values as a completion of existing proteomic data sets. As a model system we analyzed SH2-mediated interactions of the human T-cell phosphoprotein ADAP. Putative SH2 domain-containing binding partners were synthesized from a cDNA library using Expression-PCR with site-specific biotinylation in order to analyze their interaction with fluorescently labeled and in vitro phosphorylated ADAP by pull-down. On the basis of the pull-down results, selected SH2's were subjected to MST to determine Kd values. In particular, we could identify an unexpectedly strong binding of ADAP to the previously found binding partner Rasa1 of about 100 nM, while no evidence of interaction was found for the also predicted SH2D1A. Moreover, Kd values between ADAP and its known binding partners SLP-76 and Fyn were determined. Next to expanding data on ADAP suggesting promising candidates for further analysis in vivo, this work marks the first Kd values for phosphotyrosine/SH2 interactions on a phosphoprotein level.

Stabilization of peptides for intracellular applications by phosphoramidate-linked polyethylene glycol chains
Nischan, N., Chakrabarti(*), A., Serwa, R. A., Bovee-Geurts(*), P. H., Brock(*), R.; Hackenberger, C. P.
Angew Chem Int Ed Engl, 52:11920-11924
(2013)

Tags: Chemical Biology II (Hackenberger)

Alzheimer's Disease: Identification and Development of -Secretase (BACE-1) Binding Fragments and Inhibitors by Dynamic Ligation Screening (DLS)
Fernandez-Bachiller, M. I., Horatscheck, A., Lisurek, M.; Rademann, J.
Chemmedchem, 8:1041-1056
(2013)

Tags: Medicinal Chemistry (Rademann)

Abstract: The application of dynamic ligation screening (DLS), a methodology for fragment-based drug discovery (FBDD), to the aspartic protease -secretase (BACE-1) is reported. For this purpose, three new fluorescence resonance energy transfer (FRET) substrates were designed and synthesized. Their kinetic parameters (Vmax, KM, and kcat) were determined and compared with a commercial substrate. Secondly, a peptide aldehyde was designed as a chemically reactive inhibitor (CRI) based on the Swedish mutation substrate sequence. Incubation of this CRI with the protease, a FRET substrate, and one amine per well taken from an amine library, which was assembled by a maximum common substructure (MCS) approach, revealed the fragment 3-(3-aminophenyl)-2H-chromen-2-one (1) to be a competitive BACE-1 inhibitor that enhanced the activity of the CRI. Irreversibly formed fragment combination products of 1 with the initial peptide sequence were active and confirmed the targeting of the active site through the ethane-1,2-diamine isostere. Finally, structure-assisted combination of fragment 1 with secondary fragments that target the S1 site in hit optimization yielded novel, entirely fragment-based BACE-1 inhibitors with up to 30-fold improved binding affinity. Interactions with the protein were explained by molecular modeling studies, which indicate that the new fragment combinations interact with the catalytic aspartic acid dyad, as well as with the adjacent binding sites required for potency.

A Supramolecular Peptide Synthesizer
Bertran-Vicente, J.; Hackenberger, C. P. R.
Angew Chem Int Edit, 52:6140-6142
(2013)

Tags: Chemical Biology II (Hackenberger)

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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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