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Publications (8)21.97 Total impact

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    ABSTRACT: FGF-2 is an unconventionally secreted lectin that transmits proangiogenic signals through a ternary complex with high-affinity FGF receptors and heparan sulfate proteoglycans (HSPGs). Although FGF-2 signal transduction is understood in great detail, its mechanism of release from cells, which is independent of the classical secretory pathway, remains elusive. To test the hypothesis that FGF-2 secretion is linked to its cell-surface ligands, we studied FGF-2 release using mutants defective for HSPG binding and cells with impaired HSPG biosynthesis. Here, we report that a functional interaction between FGF-2 and HSPGs is required for net export of FGF-2 from mammalian cells. FGF-2 release requires extracellular, membrane-proximal HSPGs. We propose that extracellular HSPGs form a molecular trap that drives FGF-2 translocation across the plasma membrane.
    Proceedings of the National Academy of Sciences 11/2006; 103(42):15479-84. · 9.81 Impact Factor
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    ABSTRACT: The cytokine macrophage migration inhibitory factor (MIF) is inducibly secreted by immune cells and certain other cell types to critically participate in the regulation of the host immune response. However, MIF does not contain a N-terminal signal sequence and the mechanism of MIF secretion is unknown. Here we show in a model of endotoxin-stimulated THP-1 monocytes that MIF does not enter the endoplasmatic reticulum and that MIF secretion is not inhibited by monensin or brefeldin A, demonstrating that MIF secretion occurs via a non-classical export route. Glyburide and probenicide but not other typical inhibitors of non-classical protein export strongly block MIF secretion, indicating that the export pathway of MIF involves an ABCA1 transporter.
    FEBS Letters 10/2003; 551(1-3):78-86. · 3.58 Impact Factor
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    ABSTRACT: The influence of radiation-induced apoptosis on radiosensitivity was studied in a set of closely related human lymphoblastoid cell lines differing in TP53 status. The clonogenic survival of irradiated TK6 cells (expressing wild-type TP53), WTK1 cells (overexpressing mutant TP53), and TK6E6 cells (negative for TP53 owing to transfection with HPV16 E6) was assessed in relation to the induction of apoptosis and its suppression by caspase inhibition or treatment with PMA as well as after treatment with caffeine. Measurements using the alkaline comet assay and pulsed-field electrophoresis of the induction and repair of DNA strand breaks showed similar kinetics of the processing of early DNA damage in these cell lines. The cytochalasin B micronucleus assay revealed identical levels of residual damage in the first postirradiation mitosis of these cells. Abrogation of TP53-dependent apoptosis in TK6E6 cells resulted in a distinct increase in radioresistance. Further suppression of apoptosis as observed in WTK1 cells overexpressing mutant TP53 apparently was not responsible for the high radioresistance of WTK1 cells, since other means of highly efficient suppression of apoptosis (caspase inhibition or PMA treatment) increased the clonogenic survival of irradiated TK6 cells only to levels similar to those of TK6E6 cells with abrogated TP53-dependent apoptosis. Considering the similar levels of residual chromosomal damage in TK6E6 cells and WTK1 cells, a hitherto unknown mechanism of tolerance needs to be inferred for these TP53 mutant cells. This residual damage tolerance, however, appears to require an intact G2/M-phase checkpoint function since the relative radioresistance of the WTK1 cells was completely lost upon caffeine treatment, which also resulted in a failure of the TK6 and TK6E6 cells to execute apoptosis. In this situation, the cellular response seems to be dominated entirely by TP53-independent mitotic failure.
    Radiation Research 01/2003; 158(6):699-706. · 2.70 Impact Factor
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    ABSTRACT: Basic fibroblast growth factor (FGF-2) is a secretory protein that lacks a signal peptide. Consistently, FGF-2 has been shown to be secreted by an ER-Golgi-independent mechanism; however, the machinery mediating this process remains to be established at the molecular level. Here we introduce a novel experimental system based on flow cytometry that allows the quantitative assessment of non-classical FGF-2 secretion in living cells. Stable cell lines have been created by retroviral transduction that express various kinds of FGF-2-GFP fusion proteins in a doxicyclin-dependent manner. Following induction of protein expression, biosynthetic FGF-2-GFP is shown to translocate to the outer surface of the plasma membrane as determined by both fluorescence activated cell sorting (FACS) and confocal microscopy. Both N- and C-terminal GFP tagging of FGF-2 is compatible with FGF-2 export, which is shown to occur in a controlled fashion rather than through unspecific release. The experimental system described has strong implications for the identification of both FGF-2 secretion inhibitors and molecular components involved in FGF-2 secretion. In the second part of this study we made use of the FGF-2 export system described to analyze the fate of biosynthetic FGF-2-GFP following export to the extracellular space. We find that secreted FGF-2 fusion proteins accumulate in large heparan sulfate proteoglycan (HSPG)-containing protein clusters on the extracellular surface of the plasma membrane. These microdomains are shown to be distinct from caveolae-like lipid rafts known to play a role in FGF-2-mediated signal transduction. Since CHO cells lack FGF high-affinity receptors (FGFRs), it can be concluded that FGFRs mediate the targeting of FGF-2 to lipid rafts. Consistently, FGF-2-GFP-secreting CHO cells do not exhibit increased proliferation activity. Externalization and deposition of biosynthetic FGF-2 in HSPG-containing protein clusters are independent processes, as a soluble secreted intermediate was demonstrated. The balance between intracellular FGF-2 and HSPG-bound secreted FGF-2 is shown not to be controlled by the availability of cell surface HSPGs, indicating that the FGF-2 secretion machinery itself is rate-limiting.
    Journal of Cell Science 10/2002; 115(Pt 18):3619-31. · 5.88 Impact Factor
  • International Journal of Radiation Oncology Biology Physics - INT J RADIAT ONCOL BIOL PHYS. 01/2001; 51(3):229-229.
  • International Journal of Radiation Oncology Biology Physics - INT J RADIAT ONCOL BIOL PHYS. 01/2000; 48(3):279-279.
  • Andre Engling
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    ABSTRACT: The majority of secretory proteins is exported from mammalian cells by the classical secretory pathway involving subcellular compartments such as the endoplasmic reticulum (ER) and the Golgi apparatus. However, basic fibroblast growth factor (FGF2), a potent mediator of tumor-induced angiogenesis, has been shown to be secreted by a non-classical pathway that does not depend on the functions of the ER and the Golgi apparatus. The molecular characterization of the FGF2 export mechanism is not only a fundamental problem in cell biology but also of great interest for biomedical research since it may pave the way for the development of a novel class of anti-angiogenic drugs. In this thesis, a robust model system designed to quantitatively assess FGF2 secretion under various experimental conditions was developed. A retroviral expression system was established in CHO cells that allows for a stable integration of reporter constructs whose expression can be induced by doxicycline. In order to monitor expression of FGF2 reporter molecules they were constructed as GFP fusion proteins. Based on this experimental system, secretion of FGF2-GFP can be quantified by flow cytometry, confocal microscopy and biochemical methods since exported FGF2-GFP binds to cell surface heparan sulfate proteoglycans and, therefore, is accessible by membrane-impermeable tools such as antibodies and biotinylation reagents. In the second part of this thesis, a systematic mutational analysis of the FGF2 open reading frame was conducted in order to identify cis elements that direct FGF2 to its export machinery. Initial experiments revealed the identification of FGF2 mutants that are defective in binding to heparan sulfate proteoglycans. Such mutants were neither detectable on the cell surface nor in the medium of cells suggesting that the interaction of FGF2 with heparan sulfate proteoglycans does not only play a role in FGF2 signaling but also in the overall process of FGF2 externalization from mammalian cells. A collection of more than a hundred FGF2 mutants and corresponding stable cell lines described in this thesis now provide a basis for future studies in order to conduct a detailed analysis of determinants required for FGF2 secretion. Proteine mit extrazellulären Funktionen werden typischerweise über den klassischen sekretorischen Transportweg exportiert, in den subzelluläre Kompartimente wie das endoplasmatische Retikulum (ER) und der Golgi-Apparat involviert sind. Der Wachstumsfaktor Fibroblast Growth Factor 2 (FGF2) wird hingegen über einen nicht-klassischen Weg sezerniert, der nicht von der Funktion des ER und dem Golgi-Apparat abhängig ist und dessen molekulare Komponenten bisher noch völlig unbekannt sind. Die Aufklärung dieses Prozesses ist einerseits eine interessante Fragestellung in der Grundlagenforschung und andererseits von grösstem biomedizinischen Interesse, da FGF2 eine unmittelbare Rolle bei der Tumor-induzierten Angiogenese zukommt. In dieser Arbeit wurde ein robustes Modellsystem entwickelt, mit dessen Hilfe die Freisetzung von FGF2 durch Säugetierzellen exakt quantifiziert werden kann. Unter Verwendung eines retroviralen Expressionssystems wurden CHO Zellen generiert, die ein aus FGF2 und GFP bestehendes Reportermolekül in Doxicyclin-abhängiger Weise exprimieren. So konnte die Sekretion des FGF2-GFP Fusionsproteins durch Flusszytometrie, konfokale Laserscanmikroskopie und biochemische Analysen mittels Zelloberflächenbiotinylierung exakt bestimmt werden, da extrazelluläres FGF2 an Heparansulfat-Proteoglykane der Zelloberflächen bindet. Die GFP-abgeleitete Fluoreszenz des Reportermoleküls wurde zur Normalisierung der Sekretionseffizienz auf der Basis der Expressionsrate genutzt. Im zweiten Teil der vorliegenden Arbeit wurde eine systematische Analyse des offenen Leserasters von FGF2 bezüglich möglicher Exportmotive durchgeführt. Durch eine Zufallsmutagenese, den gezielten Austausch von Oberflächenresten sowie die systematische Verkürzung von N- und C-Terminus wurden mehr als 100 FGF2 Mutanten generiert und zur Etablierung stabiler Zelllinien verwendet. Mit dem oben beschriebenen System erfolgte eine vollständige Charakterisierung bezüglich Expression, Bindung an Heparansulfate und Exporteffizienz. Unter anderem wurden FGF2 Mutanten identifiziert, die die Fähigkeit zur Bindung an Heparansulfat-Proteoglykane vollständig verloren haben. Interessanterweise konnte diese FGF2 Mutanten weder an der Zelloberfläche noch im Medium detektiert werden. Hieraus wurde geschlossen, dass die funktionelle Interaktion von FGF2 mit Heparansulfat-Proteoglykanen nicht allein auf Signaltransduktionsprozesse beschränkt ist, sondern bereits eine Rolle bei der Sekretion von FGF2 spielt. Die in dieser Arbeit generierte Kollektion von FGF2 Mutanten und der zugehörigen Zelllinien bildet nun die Basis für eine detaillierte Analyse der Determinanten in FGF2, die für den Exportprozess von Bedeutung sind.