FMP Publications

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

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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.

Redox Regulation of Cell Contacts by Tricellulin and Occludin: Redox-Sensitive Cysteine Sites in Tricellulin Regulate Both Tri- and Bicellular Junctions in Tissue Barriers as Shown in Hypoxia and Ischemia
Cording, J., Günther, R., Vigolo(*), E., Tscheik, C., Winkler, L., Schlattner, I., Lorenz, D., Haseloff, R. F., Schmidt-Ott(*), K. M., Wolburg(*), H.; Blasig, I. E.
Antioxid Redox Signal, 23:1035-1049

Tags: Molecular Cell Physiology (Blasig, I.E.)

Abstract: UNLABELLED: Tight junctions (TJs) seal paracellular clefts in epithelia/endothelia and form tissue barriers for proper organ function. TJ-associated marvel proteins (TAMPs; tricellulin, occludin, marvelD3) are thought to be relevant to regulation. Under normal conditions, tricellulin tightens tricellular junctions against macromolecules. Traces of tricellulin occur in bicellular junctions. AIMS: As pathological disturbances have not been analyzed, the structure and function of human tricellulin, including potentially redox-sensitive Cys sites, were investigated under reducing/oxidizing conditions at 3- and 2-cell contacts. RESULTS: Ischemia, hypoxia, and reductants redistributed tricellulin from 3- to 2-cell contacts. The extracellular loop 2 (ECL2; conserved Cys321, Cys335) trans-oligomerized between three opposing cells. Substitutions of these residues caused bicellular localization. Cys362 in transmembrane domain 4 contributed to bicellular heterophilic cis-interactions along the cell membrane with claudin-1 and marvelD3, while Cys395 in the cytosolic C-terminal tail promoted homophilic tricellullar cis-interactions. The Cys sites included in homo-/heterophilic bi-/tricellular cis-/trans-interactions contributed to cell barrier tightness for small/large molecules. INNOVATION: Tricellulin forms TJs via trans- and cis-association in 3-cell contacts, as demonstrated electron and quantified fluorescence microscopically; it tightens 3- and 2-cell contacts. Tricellulin's ECL2 specifically seals 3-cell contacts redox dependently; a structural model is proposed. CONCLUSIONS: TAMP ECL2 and claudins' ECL1 share functionally and structurally similar features involved in homo-/heterophilic tightening of cell-cell contacts. Tricellulin is a specific redox sensor and sealing element at 3-cell contacts and may compensate as a redox mediator for occludin loss at 2-cell contacts in vivo and in vitro. Molecular interaction mechanisms were proposed that contribute to tricellulin's function. In conclusion, tricellulin is a junctional redox regulator for ischemia-related alterations.

Redox-sensitive structure and function of the first extracellular loop of the cell-cell contact protein claudin-1: lessons from molecular structure to animals
Dabrowski, S., Staat, C., Zwanziger, D., Sauer(*), R. S., Bellmann, C., Günther, R., Krause, E., Haseloff, R. F., Rittner(*), H.; Blasig, I. E.
Antioxid Redox Signal, 22:1-14

Tags: Molecular Cell Physiology (Blasig, I.E.), Mass Spectrometry (Krause, E.)

Abstract: UNLABELLED: The paracellular cleft within epithelia/endothelia is sealed by tight junction (TJ) proteins. Their extracellular loops (ECLs) are assumed to control paracellular permeability and are targets of pathogenes. We demonstrated that claudin-1 is crucial for paracellular tightening. Its ECL1 is essential for the sealing and contains two cysteines conserved throughout all claudins. AIMS: We prove the hypothesis that this cysteine motif forms a redox-sensitive intramolecular disulfide bridge and, hence, the claudin-1-ECL1 constitutes a functional structure which is associated to ECLs of this and other TJ proteins. RESULTS: The structure and function of claudin-1-ECL1 was elucidated by investigating sequences of this ECL as synthetic peptides, C1C2, and as recombinant proteins, and exhibited a beta-sheet binding surface flanked by an alpha-helix. These sequences bound to different claudins, their ECL1, and peptides with nanomolar binding constants. C-terminally truncated C1C2 (-4aaC) opened cellular barriers and the perineurium. Recombinant ECL1 formed oligomers, and bound to claudin-1 expressing cells. Oligomerization and claudin association were abolished by reducing agents, indicating intraloop disulfide bridging and redox sensitivity. INNOVATION: The structural and functional model based on our in vitro and in vivo investigations suggested that claudin-1-ECL1 constitutes a functional and ECL-binding beta-sheet, stabilized by a shielded and redox-sensitive disulfide bond. CONCLUSION: Since the beta-sheet represents a consensus sequence of claudins and further junctional proteins, a general structural feature is implied. Therefore, our model is of general relevance for the TJ assembly in normal and pathological conditions. C1C2-4aaC is a new drug enhancer that is used to improve pharmacological treatment through tissue barriers.

Identity crisis in the PMP-22/EMP/MP20/Claudin superfamily (Pfam00822)
Gehne, N., Haseloff, R. F.; Blasig, I. E.
Tissue Barriers, 3:e1089680

Tags: Molecular Cell Physiology (Blasig, I.E.)

Depletion of highly abundant proteins from human cerebrospinal fluid: a cautionary note
Günther, R., Krause, E., Schümann, M., Blasig, I. E.; Haseloff, R. F.
Mol Neurodegener, 10:53

Tags: Molecular Cell Physiology (Blasig, I.E.), Mass Spectrometry (Krause, E.)

Abstract: Affinity-based techniques, both for enrichment or depletion of proteins of interest, suffer from unwanted interactions between the bait or matrix material and molecules different from the original target. This effect was quantitatively studied by applying two common procedures for the depletion of albumin/gamma immunoglobulin to human cerebrospinal fluid. Proteins of the depleted and the column-bound fraction were identified by mass spectrometry, employing (18)O labeling for quantitation of their abundance. To different extents, the depletion procedures caused the loss of proteins previously suggested as biomarker candidates for neurological diseases. This is an important phenomenon to consider when quantifying protein levels in biological fluids.

Transmembrane proteins of the tight junctions at the blood-brain barrier: structural and functional aspects
Haseloff, R. F., Dithmer, S., Winkler, L., Wolburg, H.; Blasig, I. E.
Semin Cell Dev Biol, 38:16-25

Tags: Molecular Cell Physiology (Blasig, I.E.)

Abstract: The blood-brain barrier (BBB) is formed by microvascular endothelial cells sealed by tetraspanning tight junction (TJ) proteins, such as claudins and TAMPs (TJ-associated marvel proteins, occludin and tricellulin). Claudins are the major components of the TJs. At the BBB, claudin-5 dominates the TJs by preventing the paracellular permeation of small molecules. On the other hand, TAMPs regulate the structure and function of the TJs; tricellulin may tighten the barrier for large molecules. This review aims at integrating and summarizing the most relevant and recent work on how the BBB is influenced by claudin-1, -3, -5, -12 and the TAMPs occludin and tricellulin, all of which are four-transmembrane TJ proteins. The exact functions of claudin-1, -3, -12 and TAMPs at this barrier still need to be elucidated.

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.

Phenothiazine-derived antipsychotic drugs inhibit dynamin and clathrin-mediated endocytosis
Daniel(*), J. A., Chau(*), N., Abdel-Hamid(*), M. K., Hu(*), L., von Kleist, L., Whiting(*), A., Krishnan(*), S., Maamary(*), P., Joseph(*), S. R., Simpson(*), F., Haucke, V., McCluskey(*), A.; Robinson(*), P. J.
Traffic, 16:635-654

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Chlorpromazine is a phenothiazine-derived antipsychotic drug (APD) that inhibits clathrin-mediated endocytosis (CME) in cells by an unknown mechanism. We examined whether its action and that of other APDs might be mediated by the GTPase activity of dynamin. Eight of eight phenothiazine-derived APDs inhibited dynamin I (dynI) in the 2-12 microm range, the most potent being trifluoperazine (IC50 2.6 +/- 0.7 microm). They also inhibited dynamin II (dynII) at similar concentrations. Typical and atypical APDs not based on the phenothiazine scaffold were 8- to 10-fold less potent (haloperidol and clozapine) or were inactive (droperidol, olanzapine and risperidone). Kinetic analysis showed that phenothiazine-derived APDs were lipid competitive, while haloperidol was uncompetitive with lipid. Accordingly, phenothiazine-derived APDs inhibited dynI GTPase activity stimulated by lipids but not by various SH3 domains. All dynamin-active APDs also inhibited transferrin (Tfn) CME in cells at related potencies. Structure-activity relationships (SAR) revealed dynamin inhibition to be conferred by a substituent group containing a terminal tertiary amino group at the N2 position. Chlorpromazine was previously proposed to target AP-2 recruitment in the formation of clathrin-coated vesicles (CCV). However, neither chlorpromazine nor thioridazine affected AP-2 interaction with amphiphysin or clathrin. Super-resolution microscopy revealed that chlorpromazine blocks neither clathrin recruitment by AP-2, nor AP-2 recruitment, showing that CME inhibition occurs downstream of CCV formation. Overall, potent dynamin inhibition is a shared characteristic of phenothiazine-derived APDs, but not other typical or atypical APDs, and the data indicate that dynamin is their likely in-cell target in endocytosis.

Probing heterobivalent binding to the endocytic AP-2 adaptor complex by DNA-based spatial screening
Diezmann(*), F., von Kleist, L., Haucke, V.; Seitz(*), O.
Organic & Biomolecular Chemistry, 13:8008-8015

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: The double helical DNA scaffold offers a unique set of properties, which are particularly useful for studies of multivalency in biomolecular interactions: (i) multivalent ligand displays can be formed upon nucleic acid hybridization in a self-assembly process, which facilitates spatial screening (ii) valency and spatial arrangement of the ligand display can be precisely controlled and (iii) the flexibility of the ligand display can be adjusted by integrating nick sites and unpaired template regions. Herein we describe the use of DNA-based spatial screening for the characterization of the adaptor complex 2 (AP-2), a central interaction hub within the endocytic protein network in clathrin-mediated endocytosis. AP-2 is comprised of a core domain and two, so-called appendage domains, the alpha- and the beta 2-ear, which associate with cyto-plasmatic proteins required for the formation or maturation of clathrin/AP-2 coated pits. Each appendage domain has two binding grooves which recognize distinct peptide motives with micromolar affinity. This provides opportunities for enhanced interactions with protein molecules that contain two (or more) different peptide motives. To determine whether a particular, spatial arrangement of binding motifs is required for high affinity binding we probed the distance-affinity relationships by means of DNA-programmed spatial screening with self-assembled peptide-DNA complexes. By using trimolecular and tetra-molecular assemblies two different peptides were positioned in 2-22 nucleotide distance. The binding data obtained with both recombinant protein in well-defined buffer systems and native AP-2 in brain extract suggests that the two binding sites of the AP-2 alpha-appendage can cooperate to provide up to 40-fold enhancement of affinity compared to the monovalent interaction. The distance between the two recognized peptide motives was less important provided that the DNA duplex segments were connected by flexible, single strand segments. By contrast, the experiments with a more rigid, duplex-spaced assembly revealed marked distance dependencies. Consequences for the function of adaptor proteins are discussed.

Lipids, GTPases, and their regulators in membrane dynamics: an intracellular menage a trois
Echard(*), A.; Haucke, V.
Mol Biol Cell, 26:1012-1013

Tags: Molecular Pharmacology and Cell Biology (Haucke)

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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
+4930 94793 - 100 
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