FMP Publications

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

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Light-Dark Adaptation of Channelrhodopsin Involves Photoconversion between the all-trans and 13-cis Retinal Isomers
Bruun(*), S., Stöppler, D., Keidel(*), A., Kuhlmann(*), U., Luck(*), M., Diehl, A., Geiger, M. A., Woodmansee(*), D., Trauner(*), D., Hegemann(*), P., Oschkinat, H., Hildebrandt(*), P.; Stehfes(*)t, K.
Biochemistry, 54:5389-5400

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Channelrhodopsins (ChR) are light-gated ion channels of green algae that are widely used to probe the function of neuronal cells with light. Most ChRs show a substantial reduction in photocurrents during illumination, a process named "light adaptation". The main objective of this spectroscopic study was to elucidate the molecular processes associated with light-dark adaptation. Here we show by liquid and solid-state nuclear magnetic resonance spectroscopy that the retinal chromophore of fully dark-adapted ChR is exclusively in an all-trans configuration. Resonance Raman (RR) spectroscopy, however, revealed that already low light intensities establish a photostationary equilibrium between all-trans,15-anti and 13-cis,15-syn configurations at a ratio of 3:1. The underlying photoreactions involve simultaneous isomerization of the C(13) horizontal lineC(14) and C(15) horizontal lineN bonds. Both isomers of this DAapp state may run through photoinduced reaction cycles initiated by photoisomerization of only the C(13) horizontal lineC(14) bond. RR spectroscopic experiments further demonstrated that photoinduced conversion of the apparent dark-adapted (DAapp) state to the photocycle intermediates P500 and P390 is distinctly more efficient for the all-trans isomer than for the 13-cis isomer, possibly because of different chromophore-water interactions. Our data demonstrating two complementary photocycles of the DAapp isomers are fully consistent with the existence of two conducting states that vary in quantitative relation during light-dark adaptation, as suggested previously by electrical measurements.

Simian hemorrhagic fever virus cell entry is dependent on CD163 and uses a clathrin-mediated endocytosis-like pathway
Cai(*), Y., Postnikova(*), E. N., Bernbaum(*), J. G., Yu(*), S. Q., Mazur(*), S., Deiuliis(*), N. M., Radoshitzky(*), S. R., Lackemeyer(*), M. G., McCluskey(*), A., Robinson(*), P. J., Haucke, V., Wahl-Jensen(*), V., Bailey(*), A. L., Lauck(*), M., Friedrich(*), T. C., O'Connor(*), D. H., Goldberg(*), T. L., Jahrling(*), P. B.; Kuhn(*), J. H.
J Virol, 89:844-856

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: UNLABELLED: Simian hemorrhagic fever virus (SHFV) causes a severe and almost uniformly fatal viral hemorrhagic fever in Asian macaques but is thought to be nonpathogenic for humans. To date, the SHFV life cycle is almost completely uncharacterized on the molecular level. Here, we describe the first steps of the SHFV life cycle. Our experiments indicate that SHFV enters target cells by low-pH-dependent endocytosis. Dynamin inhibitors, chlorpromazine, methyl-beta-cyclodextrin, chloroquine, and concanamycin A dramatically reduced SHFV entry efficiency, whereas the macropinocytosis inhibitors EIPA, blebbistatin, and wortmannin and the caveolin-mediated endocytosis inhibitors nystatin and filipin III had no effect. Furthermore, overexpression and knockout study and electron microscopy results indicate that SHFV entry occurs by a dynamin-dependent clathrin-mediated endocytosis-like pathway. Experiments utilizing latrunculin B, cytochalasin B, and cytochalasin D indicate that SHFV does not hijack the actin polymerization pathway. Treatment of target cells with proteases (proteinase K, papain, alpha-chymotrypsin, and trypsin) abrogated entry, indicating that the SHFV cell surface receptor is a protein. Phospholipases A2 and D had no effect on SHFV entry. Finally, treatment of cells with antibodies targeting CD163, a cell surface molecule identified as an entry factor for the SHFV-related porcine reproductive and respiratory syndrome virus, diminished SHFV replication, identifying CD163 as an important SHFV entry component. IMPORTANCE: Simian hemorrhagic fever virus (SHFV) causes highly lethal disease in Asian macaques resembling human illness caused by Ebola or Lassa virus. However, little is known about SHFV's ecology and molecular biology and the mechanism by which it causes disease. The results of this study shed light on how SHFV enters its target cells. Using electron microscopy and inhibitors for various cellular pathways, we demonstrate that SHFV invades cells by low-pH-dependent, actin-independent endocytosis, likely with the help of a cellular surface protein.

Stonin1 mediates endocytosis of the proteoglycan NG2 and regulates focal adhesion dynamics and cell motility
Feutlinske, F., Browarski, M., Ku(*), M. C., Trnka, P., Waiczies(*), S., Niendorf(*), T., Stallcup(*), W. B., Glass(*), R., Krause, E.; Maritzen, T.
Nat Commun, 6:8535

Tags: Membrane Traffic and Cell Motility (Maritzen), Mass Spectrometry (Krause, E.)

Abstract: Cellular functions, ranging from focal adhesion (FA) dynamics and cell motility to tumour growth, are orchestrated by signals cells receive from outside via cell surface receptors. Signalling is fine-tuned by the exo-endocytic cycling of these receptors to control cellular responses such as FA dynamics, which determine cell motility. How precisely endocytosis regulates turnover of the various cell surface receptors remains unclear. Here we identify Stonin1, an endocytic adaptor of unknown function, as a regulator of FA dynamics and cell motility, and demonstrate that it facilitates the internalization of the oncogenic proteoglycan NG2, a co-receptor of integrins and platelet-derived growth factor receptor. Embryonic fibroblasts obtained from Stonin1-deficient mice display a marked surface accumulation of NG2, increased cellular signalling and defective FA disassembly as well as altered cellular motility. These data establish Stonin1 as a specific adaptor for the endocytosis of NG2 and as an important factor for FA dynamics and cell migration.

Selective inhibitors of the protein tyrosine phosphatase SHP2 block cellular motility and growth of cancer cells in vitro and in vivo
Grosskopf, S., Eckert, C., Arkona(*), C., Radetzki, S., Böhm(*), K., Heinemann(*), U., Wolber(*), G., von Kries, J. P., Birchmeier(*), W.; Rademann(*), J.
Chemmedchem, 10:815-826

Tags: Medicinal Chemistry (Rademann), Screening Unit (von Kries)

Abstract: Selective inhibitors of the protein tyrosine phosphatase SHP2 (src homology region 2 domain phosphatase; PTPN11), an enzyme that is deregulated in numerous human tumors, were generated through a combination of chemical synthesis and structure-based rational design. Seventy pyridazolon-4-ylidenehydrazinyl benzenesulfonates were prepared and evaluated in enzyme assays. The binding modes of active inhibitors were simulated in silico using a newly generated crystal structure of SHP2. The most powerful compound, GS-493 (4-(2Z)-2-[1,3-bis(4-nitrophenyl)-5-oxo-1,5-dihydro-4H-pyrazol-4-yliden]hydrazin obenzenesulfonic acid; 25) inhibited SHP2 with an IC50 value of 71+/-15 nM in the enzyme assay and was 29- and 45-fold more active toward SHP2 than against related SHP1 and PTP1B. In cell culture experiments compound 25 was found to block hepatocyte growth factor (HGF)-stimulated epithelial-mesenchymal transition of human pancreatic adenocarcinoma (HPAF) cells, as indicated by a decrease in the minimum neighbor distances of cells. Moreover, 25 inhibited cell colony formation in the non-small-cell lung cancer cell line LXFA 526L in soft agar. Finally, 25 was observed to inhibit tumor growth in a murine xenograft model. Therefore, the novel specific compound 25 strengthens the hypothesis that SHP2 is a relevant protein target for the inhibition of mobility and invasiveness of cancer cells.

Exploring monovalent and multivalent peptides for the inhibition of FBP21-tWW
Henning(*), L. M., Bhatia(*), S., Bertazzon(*), M., Marczynke(*), M., Seitz(*), O., Volkmer, R., Haag(*), R.; Freund(*), C.
Beilstein J Org Chem, 11:701-706

Tags: Peptide Synthesis (Hackenberger/Volkmer)

Abstract: The coupling of peptides to polyglycerol carriers represents an important route towards the multivalent display of protein ligands. In particular, the inhibition of low affinity intracellular protein-protein interactions can be addressed by this design. We have applied this strategy to develop binding partners for FBP21, a protein which is important for the splicing of pre-mRNA in the nucleus of eukaryotic cells. Firstly, by using phage display the optimized sequence WPPPPRVPR was derived which binds with K Ds of 80 muM and 150 microM to the individual WW domains and with a K D of 150 muM to the tandem-WW1-WW2 construct. Secondly, this sequence was coupled to a hyperbranched polyglycerol (hPG) that allowed for the multivalent display on the surface of the dendritic polymer. This novel multifunctional hPG-peptide conjugate displayed a K D of 17.6 microM which demonstrates that the new carrier provides a venue for the future inhibition of proline-rich sequence recognition by FBP21 during assembly of the spliceosome.

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.

Peptide-polymer ligands for a tandem WW-domain, an adaptive multivalent protein-protein interaction: lessons on the thermodynamic fitness of flexible ligands
Koschek, K., Durmaz(*), V., Krylova, O., Wieczorek, M., Gupta(*), S., Richter, M., Bujotzek(*), A., Fischer(*), C., Haag(*), R., Freund, C., Weber(*), M.; Rademann, J.
Beilstein J Org Chem, 11:837-847

Tags: Medicinal Chemistry (Rademann), Protein Engineering (Freund), Peptide-Lipid-Interaction/ Peptide Transport (Dathe)

Abstract: Three polymers, poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), hyperbranched polyglycerol (hPG), and dextran were investigated as carriers for multivalent ligands targeting the adaptive tandem WW-domain of formin-binding protein (FBP21). Polymer carriers were conjugated with 3-9 copies of the proline-rich decapeptide GPPPRGPPPR-NH2 (P1). Binding of the obtained peptide-polymer conjugates to the tandem WW-domain was investigated employing isothermal titration calorimetry (ITC) to determine the binding affinity, the enthalpic and entropic contributions to free binding energy, and the stoichiometry of binding for all peptide-polymer conjugates. Binding affinities of all multivalent ligands were in the microM range, strongly amplified compared to the monovalent ligand P1 with a K D > 1 mM. In addition, concise differences were observed, pHPMA and hPG carriers showed moderate affinity and bound 2.3-2.8 peptides per protein binding site resulting in the formation of aggregates. Dextran-based conjugates displayed affinities down to 1.2 microM, forming complexes with low stoichiometry, and no precipitation. Experimental results were compared with parameters obtained from molecular dynamics simulations in order to understand the observed differences between the three carrier materials. In summary, the more rigid and condensed peptide-polymer conjugates based on the dextran scaffold seem to be superior to induce multivalent binding and to increase affinity, while the more flexible and dendritic polymers, pHPMA and hPG are suitable to induce crosslinking upon binding.

Quantitative analysis of the human T cell palmitome
Morrison(*), E., Kuropka, B., Kliche(*), S., Brügger(*), B., Krause, E.; Freund(*), C.
Sci Rep, 5:11598

Tags: Mass Spectrometry (Krause, E.)

Abstract: Palmitoylation is a reversible post-translational modification used to inducibly compartmentalize proteins in cellular membranes, affecting the function of receptors and intracellular signaling proteins. The identification of protein "palmitomes" in several cell lines raises the question to what extent this modification is conserved in primary cells. Here we use primary T cells with acyl-biotin exchange and quantitative mass spectrometry to identify a pool of proteins previously unreported as palmitoylated in vivo.

Alterations in creatine metabolism observed in experimental autoimmune myocarditis using ex vivo proton magic angle spinning MRS
Muench(*), F., Retel, J., Jeuthe(*), S., (*)D, O. h.-I., van Rossum, B., Wassilew(*), K., Schmerler(*), P., Kuehne(*), T., Berger(*), F., Oschkinat, H.; Messroghli(*), D. R.
Nmr Biomed, 28:1625-1633

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Experimental autoimmune myocarditis (EAM) in rodents is an accepted model of myocarditis and dilated cardiomyopathy (DCM). Altered metabolism is thought to play an important role in the pathogenesis of DCM and heart failure (HF). Study of the metabolism may provide new diagnostic information and insights into the mechanisms of myocarditis and HF. Proton MRS ((1)H-MRS) has not yet been used to study the changes occurring in myocarditis and subsequent HF. We aimed to explore the changes in creatine metabolism using this model and compare them with the findings in healthy animals. Myocardial function of male young Lewis rats with EAM was quantified by performing left ventricular ejection fraction (LVEF) analysis in short-axis cine images throughout the whole heart. Inflammatory cellular infiltrate was assessed by immunohistochemistry. Myocardial tissue was analyzed using ex vivo proton magic angle spinning MRS ((1)H-MAS-MRS). Myocarditis was confirmed histologically by the presence of an inflammatory cellular infiltrate and CD68 positive staining. A significant increase in the metabolic ratio of Tau/tCr (taurine/total creatine) obtained by (1)H-MAS-MRS was observed in myocarditis compared with healthy controls (21 d acute EAM, 4.38 (+/-0.23); 21 d control, 2.84 (+/-0.08); 35 d chronic EAM, 4.47 (+/-0.83); 35 d control, 2.59 (+/-0.38); P < 0.001). LVEF was reduced in diseased animals (EAM, 55.2% (+/-11.3%); control, 72.6% (+/-3.8%); P < 0.01) and correlated with Tau/tCr ratio (R = 0.937, P < 0.001). Metabolic alterations occur acutely with the development of myocarditis. Myocardial Tau/tCr ratio as detected by (1)H-MRS correlates with LVEF and is able to differentiate between healthy myocardium and myocardium from rats with EAM.

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